25501, a human transferase family member and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 25501 nucleic acid molecules, which encode novel transferase family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 25501 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 25501 gene has been introduced or disrupted. The invention still further provides isolated 25501 proteins, fusion proteins, antigenic peptides and anti-25501 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/217,168, filed on Aug. 12, 2002, which claims the benefit ofU.S. Provisional Application No. 60/312,539, filed Aug. 15, 2001, thecontents of each of which are incorporated herein by this reference intheir entireties.

BACKGROUND OF THE INVENTION

In general, transferases catalyze the transfer of one molecular groupfrom a donor molecule to an acceptor molecule. Examples of suchmolecular groups include phosphate, amino, methyl, acetyl, acyl,phosphatidyl, phosphoribosyl, among other groups. The methyltransferasefamily is a large superfamily of enzymes that regulate biologicalprocesses by catalyzing the transfer of methyl groups to a wide varietyof endogenous and exogenous compounds, including DNA, RNA, proteins,hormones, neurotransmitters, drugs, and xenobiotics (Weinshilboum et al.(1999) Annu. Rev. Pharmacol. Toxicol. 39:19-52).

Methylation of DNA can play an important role in the control of geneexpression in mammalian cells. DNA methyltransferases are involved inDNA methylation and catalyze the transfer of a methyl group fromS-adenosylmethionine to cytosine residues to form 5-methylcytosine, amodified base that is found mostly at CpG sites in the genome. Thepresence of methylated CpG islands in the promoter region of genes cansuppress their expression. In different types of tumors, aberrantmethylation in the promoter region has been observed for manycancer-related genes, including tumor, metastasis or angiogenesissuppressor genes, and genes that repair DNA (Momparler and Bovenzi(2000) J. Cell Physiol. 183:145-54).

Methylation of proteins is a post-translational modification which canregulate the activity and subcellular localization of numerous proteinsand can play an important role in protein repair and reversal of proteinaging. Spontaneous protein degradation processes, including oxidation,glycation, deamidation, isomerization, and racemization which producefunctionally damaged species reflect the action of aging at themolecular level (Stadtman (1992) Science 257:1220-1224; Martin et al.(1996) Nat. Genet. 13:25-34). Methylation of these damaged proteinse.g., by methyltransferase (Shimizu et al. (2000) Arch. Biochem.Biophys. 381:225-34) can play a part in the repair pathway. Proteinmethylation is also known to be important in cellular stress responses(Desrosiers and Tanguay (1988) J. Biol. Chem. 263:4686-4692) or incellular signaling events, (Lin et al. (1996) J. Biol. Chem.271:15034-15044; Abramovich et al. (1997) EMBO J. 16:260-266).

Methylation is a process important for the catabolism of smallmolecules, such as thiol compounds and neurotransmitters. A deficiencyin thiol compound detoxification by methylation is being investigatedfor its role in rheumatoid arthritis (Waring and Emery (1993) BaillieresClin. Rheumatol. 6:337-50). Inhibition of dopamine methylation andinactivation by catechol-O-methyl transferase is a goal for therapy ofParkinson's disease (Goldstein and Lieberman (1992) Neurology 42(suppl):8-12).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of a noveltransferase family member, referred to herein as “25501”. Thetransferase molecule of the invention has characteristics of amethyltransferase or methylase. The nucleotide sequence of a cDNAencoding 25501 is shown in SEQ ID NO:1, and the amino acid sequence of a25501 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotidesequence of the coding region is depicted in SEQ ID NO:3.

Accordingly, in one aspect, the invention features a nucleic acidmolecule which encodes a 25501 protein or polypeptide, e.g., abiologically active portion of the 25501 protein. In a preferredembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2. In other embodiments, theinvention provides isolated 25501 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In still otherembodiments, the invention provides nucleic acid molecules that aresubstantially identical (e.g., naturally occurring allelic variants) tothe nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In otherembodiments, the invention provides a nucleic acid molecule whichhybridizes under a stringent hybridization condition as described hereinto a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length25501 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 25501 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded are vectors and host cells containing the 25501 nucleic acidmolecules of the invention e.g., vectors and host cells suitable forproducing polypeptides.

In another related aspect, the invention provides nucleic acid fragmentssuitable as primers or hybridization probes for the detection of25501-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a 25501 encoding nucleic acid molecule are provided.

In another aspect, the invention features 25501 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of transferase-associated or other 25501-associated disorders.In another embodiment, the invention provides 25501 polypeptides havinga 25501 activity. Preferred polypeptides are 25501 proteins including atleast one transfer domain, and, preferably, having a 25501 activity,e.g., a 25501 activity as described herein.

In other embodiments, the invention provides 25501 polypeptides, e.g., a25501 polypeptide having the amino acid sequence shown in SEQ ID NO:2;an amino acid sequence that is substantially identical to the amino acidsequence shown in SEQ ID NO:2; or an amino acid sequence encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder a stringent hybridization condition as described herein to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1or SEQ ID NO:3, wherein the nucleic acid encodes a full length 25501protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 25501 nucleic acid molecule described herein.

In a related aspect, the invention provides 25501 polypeptides orfragments operatively linked to non-25501 polypeptides to form fusionproteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically orselectively bind 25501 polypeptides.

In another aspect, the invention provides methods of screening forcompounds that modulate the expression or activity of the 25501polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating25501 polypeptide or nucleic acid expression or activity, e.g., usingthe compounds identified in the screens described herein. In certainembodiments, the methods involve treatment of conditions related toaberrant activity or expression of the 25501 polypeptides or nucleicacids, such as conditions or disorders involving aberrant or deficienttransferase function or expression. Examples of such disorders include,but are not limited to, cellular proliferative and/or differentiativedisorders, apoptotic disorders, angiogenic disorders, kidney disorders,immune e.g., inflammatory, disorders, liver disorders, neurologicaldisorders, ovary disorders, prostate disorders or metabolic disorders.

The invention also provides assays for determining the activity of orthe presence or absence of 25501 polypeptides or nucleic acid moleculesin a biological sample, including for disease diagnosis.

In a further aspect, the invention provides assays for determining thepresence or absence of a genetic alteration in a 25501 polypeptide ornucleic acid molecule, including for disease diagnosis.

In another aspect, the invention features a two dimensional array havinga plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 25501 molecule. In one embodiment,the capture probe is a nucleic acid, e.g., a probe complementary to a25501 nucleic acid sequence. In another embodiment, the capture probe isa polypeptide, e.g., an antibody specific for 25501 polypeptides. Alsofeatured is a method of analyzing a sample by contacting the sample tothe aforementioned array and detecting binding of the sample to thearray.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts a hydropathy plot of human 25501. Relatively hydrophobicresidues are shown above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) are indicated by short vertical lines just below thehydropathy trace. The numbers corresponding to the amino acid sequenceof human 25501 are indicated. Polypeptides of the invention includefragments which include: all or part of a hydrophobic sequence, e.g., asequence above the dashed line, e.g., the sequence from about amino acid258 to 267, from about 353 to 363, and from about 100 to 108 of SEQ IDNO:2; all or part of a hydrophilic sequence, e.g., a sequence below thedashed line, e.g., the sequence from about amino acid 121 to 132, fromabout 150 to 160, and from about 410 to 423 of SEQ ID NO:2; a sequencewhich includes a Cys, or a glycosylation site.

DETAILED DESCRIPTION OF THE INVENTION

The human 25501 sequence (SEQ ID NO:1), which is approximately 1971nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 1512 nucleotides,including the termination codon (nucleotides indicated as coding of SEQID NO:1; SEQ ID NO:3). The coding sequence encodes a 503 amino acidprotein (SEQ ID NO:2).

Human 25501 contains the following regions or other structural features(for general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 or the Pfam website, e.g. at Sanger Institute(pfam.sanger.ac.uk), Washington University (pfam.wustl.edu), theKarolinska Institute (pfam.cgr.kr.se) or Institut de la NationalRecherche Agronomique (pfam.jouy.inra.fr)):

-   -   a transfer domain (ProDom No. PD034341, SEQ ID NO:4) located at        about amino acid residues 280 to 411 of SEQ ID NO:2;    -   a recognition/binding domain located at about amino acid        residues 30 to 250 of SEQ ID NO:2;    -   six protein kinase C phosphorylation sites (Prosite PS00005)        located at about amino acids 47 to 49, 126 to 128, 178 to 180,        181 to 183, 206 to 208, and 210 to 212 of SEQ ID NO:2;    -   ten casein kinase II phosphorylation sites (Prosite PS00006)        located at about amino acids 10 to 13, 41 to 44, 54 to 57, 126        to 129, 179 to 182, 222 to 225, 292 to 295, 357 to 360, 431 to        434, and 456 to 459 of SEQ ID NO:2;    -   one cAMP/cGMP-dependent protein kinase phosphorylation site        (Prosite PS00004) located at about amino acids 414 to 417 of SEQ        ID NO:2;    -   one tyrosine kinase phosphorylation site (Prosite PS00007)        located at about amino acids 318 to 325 of SEQ ID NO:2;    -   one amidation site (Prosite PS00009) located at about amino        acids 377 to 380 of SEQ ID NO:2; and    -   six N-myristoylation sites (Prosite PS00008) located at about        amino acids 103 to 108, 281 to 286, 327 to 332, 337 to 342, 437        to 442, and 449 to 454 of SEQ ID NO:2.

The 25501 protein contains a significant number of structuralcharacteristics in common with members of the transferase family, inparticular, of methyltransferases. The term “family” when referring tothe protein and nucleic acid molecules of the invention means two ormore proteins or nucleic acid molecules having a common structuraldomain or motif and having sufficient amino acid or nucleotide sequencehomology as defined herein. Such family members can be naturally ornon-naturally occurring and can be from either the same or differentspecies. For example, a family can contain a first protein of humanorigin as well as other distinct proteins of human origin, oralternatively, can contain homologs of non-human origin, e.g., rat ormouse proteins. Members of a family also can have common functionalcharacteristics.

As used herein, the term “transferase” includes a protein or polypeptidewhich is capable of catalyzing the transfer of a molecular group from adonor molecule to an acceptor molecule. The methyltransferase family isa large superfamily of enzymes that regulate biological processes bycatalyzing the transfer of methyl groups from donor molecules to a widevariety of acceptor molecules, e.g. endogenous and exogenous compounds,including DNA, RNA, proteins, hormones, neurotransmitters, drugs, andxenobiotics (Weinshilboum et al. (1999) Annu. Rev. Pharmacol. Toxicol.39:19-52). In order to catalyze molecular group transfer, thetransferases must recognize or bind the group's donor then catalyze thetransfer of the group to an acceptor molecule. In the process, thetransferase itself can become an intermediate acceptor molecule, e.g.,the alkylation of an active site cysteine in O(6)-alkylguanine-DNAalkyltransferase (Daniels and Tainer (2000) Mutat. Res. 460:151-163).Members of a transferase family of proteins typically are cytoplasmic ornuclear proteins. Transferases, e.g. methyltransferases typicallyinclude conserved motifs, including at least one Prositemethyltransferase signature sequence, e.g. PS01261, PS00092, or PS01184.The 25501 molecules of the invention include regions homologous to thesemotifs.

A 25501 polypeptide can include a “transfer domain” or a regionhomologous with a “transfer domain”.

As used herein, the term “transfer domain” includes an amino acidsequence of about 50 to 250 amino acid residues in length and includesone, two, preferably three sequences homologous to the Prosite methylaseor methyltransferase signature sequences PS01261, PS00092, and PS01184.Preferably, a transfer domain includes at least about 100 to 200 aminoacids, more preferably about 120 to 150 amino acid residues, or about130 to 140 amino acids and includes one, two, preferably three sequenceshomologous to Prosite methylase or methyltransferase signature sequencesPS01261, PS00092, and PS01184. Preferably the Prosite sequences arearranged in the following order, first the PS01261, second the PS00092,third the PS01184 and are spaced about sixty amino acids or less fromeach other. Preferably a transfer domain catalyzes the transfer of agroup, e.g. a methyl group from a donor to an acceptor molecule. Thetransfer domain of 25501 can be found at about amino acid residues 280to 411 of SEQ ID NO:2).

A sequence similar to the Prosite sequence PS01261, the putative RNAmethylase family UPF0020 signature, D-P-[LIVMF]-C-G-[ST]-G-x(3)-[LI]-E(SEQ ID NO:6) can be found in human 25501 at about amino acid residues304 to 315 of SEQ ID NO:2, except an L replaces the [ST]. A sequencesimilar to the Prosite sequence PS00092, the N-6 adenine-specific DNAmethylase signature, [LIVMAC]-[LIVFYWA]-x-[DN]-P-P-[FYW] (SEQ ID NO:7)can be found in human 25501 at about amino acid residues 371 to 377 ofSEQ ID NO:2, except an I replaces the first P. A sequence similar to theProsite sequence PS01184, the ubiE/COQ5 methyltransferase familysignature 2, R-V-[LIVM]-K-[PV]-[GM]-G-x-[LIVMF]-x(2)-[LIVM]-E-x-S (SEQID NO:8) can be found in human 25501 at about amino acid residues 396 to409 of SEQ ID NO:2, except an H replaces the K and the last threeresidues are L-S-E instead of E-x-S. In the above conserved signaturesequences, and other motifs or signature sequences described herein, thestandard IUPAC one-letter code for the amino acids is used. Each elementin the pattern is separated by a dash (−); square brackets ([ ])indicate the particular residues that are accepted at that position; xindicates that any residue is accepted at that position; and numbers inparentheses (O) indicate the number of residues represented by theaccompanying amino acid.

The transfer domain of the human 25501 protein is homologous, e.g., atleast about 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, or 41% identical to the ProDom family PD034341 (“VNG2242CY71F9AL.1 MTH724 PH0338 AF1257 MJ0710 APE1835”) domain (ProDomainRelease 2001.1; for ProDom information, refer to Institut National de laRecherche Agronomique (INRA)/Central National de la RechercheScientifique (CNRA), Toulouse, France). The ProDom PD034341 domain andcan include one, two, preferably three Prosite methylase ormethyltransferase signature sequences or sequences homologous to thesesequences spaced sixty amino acids or less apart. A GAP alignment of thetransfer domain (amino acids 280 to 411 of SEQ ID NO:2) of human 25501with amino acid residues 1 to 133 of the 172 amino acid PD034341 domainconsensus sequence (SEQ ID NO:4), derived from a BLAST search modelresults in 32% identity (as calculated from the blosum62 matrix).

In a preferred embodiment, a 25501 polypeptide or protein has a“transfer domain” or a region which includes at least about 100 to 200more preferably about 120 to 150 or 130 to 140 amino acid residues andhas at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a“transfer domain,” e.g., the transfer domain of human 25501 (e.g.,residues 280 to 411 of SEQ ID NO:2).

Regions similar to the transfer domain are found in other proteins. Forexample, a transfer domain can be found in MGC:2454 (SEQ ID NO:5,accession number 13278783 in GenPept; corresponding to number BC004163in GenBank). MGC:2454 is homologous to the 25501 protein in SEQ ID NO:2.An alignment of the 25501 protein with MGC:2454 results in about 94%overall sequence identity between the two sequences. Sequence identityof 100% can be found in regions beginning about amino acid 1 to 473 ofMGC:2454 (SEQ ID NO:5) with amino acids about 31 to 503 of 25501, SEQ IDNO:2 (as calculated in matblas from the blosum62.iij matrix).

To make the determination that the “transfer” domain in a 25501 proteinsequence or a polypeptide or protein of interest has a particularprofile, the amino acid sequence of the protein can be searched againsta database of domains, e.g., the ProDom database (Corpet et al. (1999),Nucl. Acids Res. 27:263-267). The ProDom protein domain databaseconsists of an automatic compilation of homologous domains. Currentversions of ProDom are built using recursive PSI-BLAST searches(Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al.(1999) Computers and Chemistry 23:333-340) of the SWISS-PROT 38 andTREMBL protein databases. The database automatically generates aconsensus sequence for each domain. A BLAST search was performed againstthe database resulting in the PD034341 profile of the “transfer” domainin the amino acid sequence of human 25501 at about residues 280 to 411of SEQ ID NO:2.

A 25501 molecule can further include a recognition/binding domain orregions homologous with a “recognition/binding domain.” As used herein,the “recognition/binding domain” includes an amino acid sequence ofabout 100 to 350 amino acid residues in length and whose secondarystructure is characterized by a high alpha helical content. Table 1,below, illustrates the prediction of the likelihood of amino acidresidues from this region of 25501 to belong to an element of secondarystructure by two prediction methods. TABLE 1 Secondary StructurePrediction of Amino Acid Residues 117 to 198 of SEQ ID NO: 2 A B C D 30V H H 31 M H H 32 R H H 33 E H H 34 V H H 35 R H H 36 A H H 37 R H H 38L H H 39 A H H 40 A H H 41 T H H 42 Q H H 43 V H H 44 E H H 45 Y . H 46I . T 47 S t T 48 G t T 49 K . T 50 V . B 51 F . B 52 F . B 53 T . B 54T t B 55 C t H 56 S T H 57 D T H 58 L . H 59 N . H 60 M H H 61 L H H 62K H H 63 K H H 64 L H H 65 K H H 66 S H H 67 A H H 68 E H H 69 R B H 70L B H 71 F B H 72 L B H 73 L B H 74 I B H 75 K . H 76 K . H 77 Q B B 78F B B 79 P B B 80 L B B 81 I B B 82 I B B 83 S . B 84 S . . 85 V . . 86S . . 87 K h . 88 G h . 89 K h . 90 I h . 91 F h B 92 N h B 93 E h B 94M b B 95 Q b . 96 R b . 97 L b . 98 I b . 99 N b . 100 E t . 101 D t .102 P T T 103 G T T 104 S T T 105 W B . 106 L B . 107 N B . 108 A B .109 I B . 110 S B . 111 I B . 112 W B H 113 K B H 114 N B H 115 L B H116 L B H 117 E H H 118 L H H 119 D H H 120 A H H 121 K H H 122 K H H123 E H H 124 K H H 125 L H H 126 S H H 127 Q H H 128 R H H 129 D t H130 D T H 131 N T H 132 Q H H 133 L H . 134 K H . 135 R H . 136 K H .137 V H . 138 G H H 139 E H H 140 N H H 141 E H H 142 I H H 143 I H H144 A H H 145 K H H 146 K H H 147 L H H 148 K H H 149 I H H 150 E H H151 Q H H 152 M H H 153 Q H H 154 K H H 155 I H H 156 E H H 157 E H H158 N . H 159 R T H 160 D T H 161 C t H 162 Q H H 163 L H H 164 E H H165 K H H 166 Q H H 167 I H H 168 K H H 169 E H H 170 E H H 171 T H H172 L H H 173 E H H 174 Q H H 175 R H H 176 D H H 177 F H H 178 T H H179 T H . 180 K H . 181 S H . 182 E H . 183 K H H 184 F H H 185 Q H H186 E H H 187 E H H 188 E H H 189 F H H 190 Q t H 191 N t H 192 D H H193 I H H 194 E H H 195 K H H 196 A H H 197 I H H 198 D H H 199 T t .200 H t . 201 N t . 202 Q T T 203 N T T 204 D t T 205 L B T 206 T B T207 F B T 208 R B T 209 V B T 210 S . T 211 C t T 212 R T T 213 C T T214 S T T 215 G T T 216 T . T 217 I . . 218 G . . 219 K H . 220 A H .221 F H H 222 T H H 223 A H H 224 Q H H 225 E H H 226 V H H 227 G . H228 K . H 229 V . H 230 I . H 231 G . H 232 I h H 233 A h H 234 I h H235 M h H 236 K h H 237 H h H 238 F h T 239 G h T 240 W h T 241 K h .242 A h . 243 D h . 244 L h . 245 R t . 246 N t . 247 P t . 248 Q t .249 L . B 250 E . BLegend:Column A represents the position in 25501 for each amino acid in thedomain,column B depicts the amino acid at that position,column C depicts the Chou-Fasman secondary structure prediction for thatamino acid (Chou and Fasman (1974) Biochemistry 13: 222-244), andcolumn D depicts the Garnier-Osguthorpe-Robson secondary structureprediction for that amino acid (Garnier et al. (1978) J. Mol. Biol. 120:97-120).Capital letters represent strong predictions,lower case letters represent weaker and“.” represents random coil or no prediction of one of the followingmotifs,“H” or “h” for alpha helix,“B” or “b” for beta sheet strand, and“T” or “t” for a turn.

As shown in Table 1, the prediction methods agree that the majority ofresidues in this region, in particular, residues 117 to 198 of SEQ IDNO:2, can form alpha helices. Proteins can use alpha helices torecognize and bind nucleic acid molecules. For example, thehelix-turn-helix DNA binding domain is involved in a variety ofprotein-DNA interactions (Wintjens and Rooman (1996) J. Mol. Biol.262:294-313), with variations in additional helices and helixarrangements distinguishing protein families from one another. Proteinscan use alpha helices to determine the specificity of ligandinteractions. For example, amino acid residues on helices in the ligandbinding pocket of steroid receptors allow the discrimination betweendifferent steroid hormones (Ekena et al. (1998) J. Biol. Chem.273:693-699).

In a preferred embodiment, a 25501 polypeptide or protein has a“recognition/binding domain” or a region which includes at least about150 to 300 more preferably about 180 to 260 or 210 to 230 amino acidresidues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%homology with a “recognition/binding domain,” e.g., therecognition/binding domain of human 25501 (e.g., residues 30 to 250 ofSEQ ID NO:2).

To identify the presence of a “recognition/binding” domain in a 25501protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be analyzed by a secondary structure prediction methodthat predicts the secondary structure of proteins based on thecharacteristics of each amino acid (Chou and Fasman (1974) Biochemistry13:222-244 and Garnier et al. (1978) J. Mol. Biol. 120:97-120).

A 25501 family member can include at least one transfer domain. A 25501family member also can include at least one recognition/binding domain.Furthermore, a 25501 family member can include at least one, two, three,four, five preferably six protein kinase C phosphorylation sites(Prosite PS00005); at least one, two, three, four, five, six, seven,eight, nine and preferably ten casein kinase II phosphorylation sites(Prosite PS00006); at least one tyrosine kinase phosphorylation site(Prosite PS00007); at least one cAMP/cGMP protein kinase phosphorylationsites (Prosite PS00004); at least one amidation site (Prosite PS00009);and at least one, two, three, four, five preferably six N-myristoylationsites (Prosite PS00008).

As the 25501 polypeptides of the invention can modulate 25501-mediatedactivities, they can be useful for developing novel diagnostic andtherapeutic agents for transferase-associated or other 25501-associateddisorders, as described below.

As used herein, a “transferase-associated activity” includes an activitywhich involves a transfer function, e.g. the transfer of a group, e.g. amethyl group from a donor molecule to an acceptor molecule. Thisfunction is implicated in a wide range of cell activities, including,but not limited to cell growth and cell processes, e.g., the regulationof cell proliferation, differentiation, migration, protein transport,gene expression, and/or intra- or intercellular signaling, andapoptosis. Members of the family can play a role in cancer,developmental syndromes, such as Fragile X and Rett (El-Osta and Wolf(2000) Gene Expr. 9:63-75), neurodegenerative disorders such asAlzheimer's disease (Shimizu et al. (2000) Arch. Biochem. Biophys.381:225-34), and Parkinson's disease (Goldstein and Lieberman (1992)Neurology 42 (suppl4):8-12), and inflammatory disorders such asrheumatoid arthritis (Waring and Emery (1992) Baillieres Clin.Rheumatol. 6:337-50).

As used herein, a “25501 activity”, “biological activity of 25501” or“functional activity of 25501”, refers to an activity exerted by a 25501protein, polypeptide or nucleic acid molecule on e.g., a25501-responsive cell or on a 25501 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 25501activity is a direct activity, such as an association with a 25501target molecule. A “target molecule” or “binding partner” is a moleculewith which a 25501 protein binds or interacts in nature. In an exemplaryembodiment, 25501 is a transferase, e.g., a methyltransferase, and thushas the ability to bind to, or interact with, a substrate or targetmolecule, e.g., a nucleic acid molecule (e.g. DNA or RNA), a smallorganic molecule (e.g., a hormone, a neurotransmitter or a coenzyme), ora protein; and/or the ability to transfer a group, e.g. a methyl groupfrom a donor to an acceptor molecule, e.g. the substrate or targetmolecule.

A 25501 activity can also be an indirect activity, e.g., a cellularsignaling activity mediated by interaction of the 25501 protein with a25501 receptor. Based on the above-described sequence structures andsimilarities to molecules of known function, the 25501 molecules of thepresent invention can have similar biological activities as transferasefamily members. For example, the 25501 proteins of the present inventioncan have one or more of the following activities: (1) the ability tointeract with a 25501 substrate or target molecule (e.g., a non-25501protein); (2) the ability to convert a 25501 substrate or targetmolecule to a product (e.g., transfer of a methyl group to or from thesubstrate or target molecule); (3) the ability to interact with and/ormethyl transfer to a second non-25501 target molecule e.g., a nucleicacid molecule (e.g., DNA or RNA), a small organic molecule (e.g., ahormone, neurotransmitter or a coenzyme) or a protein; (4) the abilityto regulate substrate or target molecule activity; (6) the ability tomodulate intra- or intercellular signaling and/or gene transcription(e.g., either directly or indirectly); (7) the ability to modulatecellular targeting and/or transport of proteins; (8) the ability tomodulate cellular proliferation, growth, or differentiation; (9) theability to modulate cell migration and/or (10) the ability to modulateapoptosis.

The 25501 molecules of the invention can modulate the activities ofcells in tissues where they are expressed. For example, 25501 mRNA isexpressed in brain, in particular the astrocytes, which provide physicaland biochemical support for neurons and interact with capillaryendothelial cells to form the blood-brain barrier. 25501 mRNA also canbe found in the ovary and prostate epithelium. 25501 mRNA also isexpressed in tissues undergoing large amounts of growth, differentiationand angiogenesis such as fetal and neonatal kidney, fetal heart andfetal adrenal gland. 25501 mRNA also is expressed in cancerous tissue,especially malignant tumors, such as Wilm's tumor, lung tumor, colontumor, metastases of colon tumor in the liver, metastases of prostatetumor in the liver, metastases of breast tumors in the lung and brain.Accordingly, the 25501 molecules of the invention can act as noveldiagnostic targets or therapeutic agents for neurological disorders,ovarian disorders, prostate disorders, or proliferative and/ordifferentiative disorders or other transferase disorders.

The 25501 molecules can be used to treat neurological disorders in partbecause the 25501 mRNA is expressed in the brain and astrocytes.Neurological disorders include CNS, cognitive and neurodegenerativedisorders, Examples of neurological disorders include, but are notlimited to, autonomic function disorders such as hypertension and sleepdisorders, and neuropsychiatric disorders, such as depression,schizophrenia, schizoaffective disorder, Korsakoff's psychosis,alcoholism, anxiety disorders, or phobic disorders; learning or memorydisorders, e.g., amnesia or age-related memory loss, attention deficitdisorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. Suchneurological disorders include, for example, disorders involvingneurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer's disease and Pick'sdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson's disease (paralysisagitans) and other Lewy diffuse body diseases, progressive supranuclearpalsy, corticobasal degenration, multiple system atrophy, includingstriatonigral degenration, Shy-Drager syndrome, and olivopontocerebellaratrophy, and Huntington's disease, senile dementia, Gilles de laTourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

The 25501 molecules can be used to treat ovarian disorders in partbecause the 25501 mRNA is expressed in the ovary. Disorders involvingthe ovary include, for example, polycystic ovarian disease,Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

The 25501 molecules can be used to treat prostate disorders in partbecause the 25501 mRNA is expressed in prostate epithelial cells.Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

The 25501 molecules can be used to treat proliferative and/ordifferentiative disorders in part because the 25501 mRNA is expressed incolon tumor, metastatic tumors of colon to liver, metastatic tumors ofprostate to liver, metastatic tumors of breast to lung and brain, Wilm'stumor and lung tumor. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma,metastatic disorders or hematopoietic neoplastic disorders, e.g.,leukemias. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of prostate, colon,lung, breast and liver origin.

As used herein, the term “cancer” (also used interchangeably with theterms, “hyperproliferative” and “neoplastic”) refers to cells having thecapacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Cancerous diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, e.g., malignant tumor growth, or may becategorized as non-pathologic, i.e., a deviation from normal but notassociated with a disease state, e.g., cell proliferation associatedwith wound repair. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. The term “cancer” includes malignanciesof the various organ systems, such as those affecting lung, breast,thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as wellas adenocarcinomas which include malignancies such as most coloncancers, renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus. The term “carcinoma” is art recognized andrefers to malignancies of epithelial or endocrine tissues includingrespiratory system carcinomas, gastrointestinal system carcinomas,genitourinary system carcinomas, testicular carcinomas, breastcarcinomas, prostatic carcinomas, endocrine system carcinomas, andmelanomas. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The term“carcinoma” also includes carcinosarcomas, e.g., which include malignanttumors composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumor cells form recognizable glandular structures. Theterm “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

The 25501 molecules of the invention can be used to monitor, treatand/or diagnose a variety of proliferative disorders. Such disordersinclude hematopoietic neoplastic disorders. As used herein, the term“hematopoietic neoplastic disorders” includes diseases involvinghyperplastic/neoplastic cells of hematopoietic origin, e.g., arisingfrom myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Preferably, the diseases arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991)Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include,but are not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

The 25501 protein, fragments thereof, and derivatives and other variantsof the sequence in SEQ ID NO:2 thereof are collectively referred to as“polypeptides or proteins of the invention” or “25501 polypeptides orproteins”. Nucleic acid molecules encoding such polypeptides or proteinsare collectively referred to as “nucleic acids of the invention” or“25501 nucleic acids.”

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleicacid molecules which are separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Forexample, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a 25501protein, preferably a mammalian 25501 protein, and can further includenon-coding regulatory sequences, and introns.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.In one embodiment, the language “substantially free” means preparationof 25501 protein having less than about 30%, 20%, 10% and morepreferably 5% (by dry weight), of non-25501 protein (also referred toherein as a “contaminating protein”), or of chemical precursors ornon-25501 chemicals. When the 25501 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.The invention includes isolated or purified preparations of at least0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of 25501 (e.g., the sequence of SEQ ID NO:1or 3) without abolishing or more preferably, without substantiallyaltering a biological activity, whereas an “essential” amino acidresidue results in such a change. For example, amino acid residues thatare conserved among the polypeptides of the present invention, e.g.,those present in the transfer domain, are predicted to be particularlyunamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a 25501 protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a 25501 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor 25501 biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1 or SEQ ID NO:3, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined.

As used herein, a “biologically active portion” of a 25501 proteinincludes a fragment of a 25501 protein which participates in aninteraction between a 25501 molecule and a non-25501 molecule.Biologically active portions of a 25501 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 25501 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include fewer amino acids than thefull length 25501 protein, and exhibit at least one activity of a 25501protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the 25501 protein, e.g.,transferase, e.g. methyltransferase activity in which it binds to orinteracts in nature with a substrate or target molecule, e.g., a nucleicacid molecule (e.g. DNA or RNA), a small organic molecule (e.g., ahormone, a neurotransmitter or a coenzyme), or a protein; transfers agroup, e.g. a methyl group from a donor to an acceptor molecule, e.g.the substrate or target molecule; and/or plays a role in cell growth andcell processes. A biologically active portion of a 25501 protein can bea polypeptide which is, for example, 10, 25, 50, 100, 200 or more aminoacids in length. Biologically active portions of a 25501 protein can beused as targets for developing agents which modulate a 25501 mediatedactivity, e.g., transferase activity e.g. methyltransferase activity inwhich it binds to, or interacts with, a substrate or target molecule,e.g., a nucleic acid molecule (e.g. DNA or RNA), a small organicmolecule (e.g., a hormone, a neurotransmitter or a coenzyme), or aprotein; transfers a group, e.g. a methyl group from a donor to anacceptor molecule, e.g. the substrate or target molecule; and/or plays arole in cell growth and cell processes.

Calculations of homology or sequence identity (the terms “homology” and“identity” are used interchangeably herein) between sequences areperformed as follows:

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 95% or 100% of the length of thereference sequence (e.g., when aligning a second sequence to the 25501amino acid sequence of SEQ ID NO:2 having 503 amino acid residues, atleast [30%] 150, preferably at least [40%] 201, more preferably at least[50%] 251, even more preferably at least [60%] 300, and even morepreferably at least [70%] 352, [80%] 402, [90%] 452, [95%] 477, [96%]482, [97%] 487, [98%] 492, or [99%] 497 amino acid residues arealigned). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package (available at the bioinformaticspage of the website maintained by Accelrys, Inc., San Diego, Calif.,USA), using either a Blossum 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package, using a NWSgapdna.CMP matrix and a gap weightof 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) are a Blossum 62 scoring matrixwith a gap penalty of 12, a gap extend penalty of 4, and a frameshiftgap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Meyers and Miller ((1989) CABIOS,4:11-17) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 25501 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 25501 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (accessible at the website maintained by National Center forBiotechnology Information, Bethesda, Md., USA).

Particular 25501 polypeptides of the present invention have an aminoacid sequence substantially identical to the amino acid sequence of SEQID NO:2. In the context of an amino acid sequence, the term“substantially identical” is used herein to refer to a first amino acidthat contains a sufficient or minimum number of amino acid residues thatare i) identical to, or ii) conservative substitutions of aligned aminoacid residues in a second amino acid sequence such that the first andsecond amino acid sequences can have a common structural domain and/orcommon functional activity. For example, amino acid sequences thatcontain a common structural domain having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 are termedsubstantially identical.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or 3 are termedsubstantially identical.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

“Subject”, as used herein, can refer to a mammal, e.g., a human, or toan experimental or animal or disease model. The subject can also be anon-human animal, e.g., a horse, cow, goat, or other domestic animal.

A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a 25501 polypeptide described herein, e.g., afull length 25501 protein or a fragment thereof, e.g., a biologicallyactive portion of 25501 protein. Also included is a nucleic acidfragment suitable for use as a hybridization probe, which can be used,e.g., to identify a nucleic acid molecule encoding a polypeptide of theinvention, 25501 mRNA, and fragments suitable for use as primers, e.g.,PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, or a portion ofany of this nucleotide sequence. In one embodiment, the nucleic acidmolecule includes sequences encoding the human 25501 protein (i.e., “thecoding region” of SEQ ID NO:1, as shown in SEQ ID NO:3), as well as 5′untranslated sequences (nucleotides 1 to 15 of SEQ ID NO:1) and 3′untranslated sequences (nucleotides 1528 to 1971 of SEQ ID NO:1).Alternatively, the nucleic acid molecule can include only the codingregion of SEQ ID NO:1 (e.g., SEQ ID NO:3) and, e.g., no flankingsequences which normally accompany the subject sequence. In anotherembodiment, the nucleic acid molecule encodes a sequence correspondingto a fragment of the protein from about amino acid residues 280 to 411,or a fragment thereof, e.g. about amino acid residues 280 to 320, 320 to360 or 360 to 411 of SEQ ID NO:2.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a portion ofany of these nucleotide sequences. In other embodiments, the nucleicacid molecule of the invention is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:1 or 3, therebyforming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the entire length of the nucleotide sequenceshown in SEQ ID NO:1 or SEQ ID NO:3, or a portion, preferably of thesame length, of any of these nucleotide sequences.

25501 Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1 or 3. For example, such anucleic acid molecule can include a fragment which can be used as aprobe or primer or a fragment encoding a portion of a 25501 protein,e.g., an immunogenic or biologically active portion of a 25501 protein.A fragment can comprise those nucleotides of SEQ ID NO:1, which encode atransfer domain of human 25501. The nucleotide sequence determined fromthe cloning of the 25501 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other 25501family members, or fragments thereof, as well as 25501 homologs, orfragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein or fragments thereof, particularlyfragments thereof which are at least 130, 140, or 210 amino acids inlength. Fragments also include nucleic acid sequences corresponding tospecific amino acid sequences described above or fragments thereof.Nucleic acid fragments should not to be construed as encompassing thosefragments that may have been disclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 25501 nucleic acid fragment caninclude a sequence corresponding to a transfer domain, as describedherein.

25501 probes and primers are provided. Typically a probe/primer is anisolated or purified oligonucleotide. The oligonucleotide typicallyincludes a region of nucleotide sequence that hybridizes under stringentconditions to at least about 7, 12 or 15, preferably about 20 or 25,more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutivenucleotides of a sense or antisense sequence of SEQ ID NO:1 or SEQ IDNO:3, or of a naturally occurring allelic variant or mutant of SEQ IDNO:1 or SEQ ID NO:3.

In a preferred embodiment the nucleic acid is a probe which is at least5 or 10, and less than 200, more preferably less than 100, or less than50, base pairs in length. It should be identical, or differ by 1, orless than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes:

-   -   a transfer domain from about amino acid 280 to 411 of SEQ ID        NO:2, or a recognition/binding domain from about amino acid 30        to 250 of SEQ ID NO:2.

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 25501 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differ by one base from asequence disclosed herein or from a naturally occurring variant. Forexample, primers suitable for amplifying all or a portion of any of thefollowing regions are provided: a transfer domain from about amino acid280 to 411 of SEQ ID NO:2.

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment can encode the N-terminal 30 amino acids of SEQID NO:2.

A nucleic acid fragment encoding a “biologically active portion of a25501 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1 or 3, which encodes a polypeptidehaving a 25501 biological activity (e.g., the biological activities ofthe 25501 proteins are described herein), expressing the encoded portionof the 25501 protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the 25501 protein. Forexample, a nucleic acid fragment encoding a biologically active portionof 25501 includes a transfer domain, e.g., amino acid residues about 280to 411 of SEQ ID NO:2. A nucleic acid fragment encoding a biologicallyactive portion of a 25501 polypeptide, can comprise a nucleotidesequence which is greater than 390, 420, 630 or more nucleotides inlength.

In preferred embodiments, a nucleic acid includes a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900 or more nucleotides in lengthand hybridizes under stringent hybridization conditions to a nucleicacid molecule of SEQ ID NO:1 or SEQ ID NO:3.

25501 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. Suchdifferences can be due to degeneracy of the genetic code (and result ina nucleic acid which encodes the same 25501 proteins as those encoded bythe nucleotide sequence disclosed herein. In another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence which differs,by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residuesthat shown in SEQ ID NO:2. If alignment is needed for this comparisonthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one codon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO:1 or 3, e.g., as follows: by at least one but less than 10, 20, 30,or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of thenucleotides in the subject nucleic acid. If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2 or a fragment of this sequence. Suchnucleic acid molecules can readily be identified as being able tohybridize under stringent conditions, to the nucleotide sequence shownin SEQ ID NO 2 or a fragment of the sequence. Nucleic acid moleculescorresponding to orthologs, homologs, and allelic variants of the 25501cDNAs of the invention can further be isolated by mapping to the samechromosome or locus as the 25501 gene.

Preferred variants include those that are correlated with transferase,e.g. methyltransferase activity in which 25501 binds to or interacts innature with a substrate or target molecule, e.g., a nucleic acidmolecule (e.g. DNA or RNA), a small organic molecule (e.g., a hormone, aneurotransmitter or a coenzyme), or a protein; transfers a group, e.g. amethyl group from a donor to an acceptor molecule, e.g. the substrate ortarget molecule; and/or plays a role in cell growth and cell processes.

Allelic variants of 25501, e.g., human 25501, include both functionaland non-functional proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the 25501 protein within apopulation that maintain the ability to bind to or interact in naturewith a substrate or target molecule, e.g., a nucleic acid molecule (e.g.DNA or RNA), a small organic molecule (e.g., a hormone, aneurotransmitter or a coenzyme), or a protein; the ability to transfer agroup, e.g. a methyl group from a donor to an acceptor molecule, e.g.the substrate or target molecule; and/or the ability to play a role incell growth and cell processes. Functional allelic variants willtypically contain only conservative substitution of one or more aminoacids of SEQ ID NO:2, or substitution, deletion or insertion ofnon-critical residues in non-critical regions of the protein.Non-functional allelic variants are naturally-occurring amino acidsequence variants of the 25501, e.g., human 25501, protein within apopulation that do not have the ability to bind to or interact in naturewith a substrate or target molecule, e.g., a nucleic acid molecule (e.g.DNA or RNA), a small organic molecule (e.g., a hormone, aneurotransmitter or a coenzyme), or a protein, the ability to transfer agroup, e.g. a methyl group from a donor to an acceptor molecule, e.g.the substrate or target molecule; and/or play a role in cell growth andcell processes. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, or a substitution,insertion, or deletion in critical residues or critical regions of theprotein.

Moreover, nucleic acid molecules encoding other 25501 family membersand, thus, which have a nucleotide sequence which differs from the 25501sequences of SEQ ID NO:1 or SEQ ID NO:3 are intended to be within thescope of the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified 25501 NucleicAcid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to 25501. An “antisense” nucleic acid caninclude a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire25501 coding strand, or to only a portion thereof (e.g., the codingregion of human 25501 corresponding to SEQ ID NO:3). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding 25501 (e.g., the 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of 25501 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of 25501 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 25501 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a 25501 protein to thereby inhibitexpression of the protein, e.g., by inhibiting transcription and/ortranslation. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically or selectively bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a 25501-encodingnucleic acid can include one or more sequences complementary to thenucleotide sequence of a 25501 cDNA disclosed herein (i.e., SEQ ID NO:1or SEQ ID NO:3), and a sequence having known catalytic sequenceresponsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoffand Gerlach (1988) Nature 334:585-591). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a 25501-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,25501 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Barteland Szostak (1993) Science 261:1411-1418.

25501 gene expression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the 25501 (e.g., the 25501promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 25501 gene in target cells. See generally,Helene (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15. Thepotential sequences that can be targeted for triple helix formation canbe increased by creating a so-called “switchback” nucleic acid molecule.Switchback molecules are synthesized in an alternating 5′-3′,3′-5′manner, such that they base pair with first one strand of a duplex andthen the other, eliminating the necessity for a sizeable stretch ofeither purines or pyrimidines to be present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A 25501 nucleic acid molecule can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup et al. (1996) supra;Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of 25501 nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of 25501 nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup et al. (1996) supra; Perry-O Keefe supra).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to a25501 nucleic acid of the invention, two complementary regions onehaving a fluorophore and one a quencher such that the molecular beaconis useful for quantitating the presence of the 25501 nucleic acid of theinvention in a sample. Molecular beacon nucleic acids are described, forexample, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al.,U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Isolated 25501 Polypeptides

In another aspect, the invention features, an isolated 25501 protein, orfragment, e.g., a biologically active portion, for use as immunogens orantigens to raise or test (or more generally to bind) anti-25501antibodies. 25501 protein can be isolated from cells or tissue sourcesusing standard protein purification techniques. 25501 protein orfragments thereof can be produced by recombinant DNA techniques orsynthesized chemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpost-translational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepost-translational modifications present when the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of post-translational modifications, e.g., glycosylation orcleavage, present in a native cell.

In a preferred embodiment, a 25501 polypeptide has one or more of thefollowing characteristics:

-   -   it has the ability to bind to or interact in nature with a        substrate or target molecule, e.g., a nucleic acid molecule        (e.g. DNA or RNA), a small organic molecule (e.g., a hormone, a        neurotransmitter or a coenzyme), or a protein;    -   the ability to transfer a group, e.g. a methyl group from a        donor to an acceptor molecule, e.g. the substrate or target        molecule;    -   the ability to modulate intra- or intercellular signaling and/or        gene transcription (e.g., either directly or indirectly);    -   the ability to modulate cellular proliferation, growth, or        differentiation;    -   it has a molecular weight, e.g., a deduced molecular weight,        preferably ignoring any contribution of post translational        modifications, amino acid composition or other physical        characteristic of a 25501 polypeptide, e.g., a polypeptide of        SEQ ID NO:2;    -   it has an overall sequence similarity of at least 60%,        preferably at least 70%, more preferably at least 80%, 90%, 95%,        96%, 97%, 98%, 99% or more with a polypeptide of SEQ ID NO:2;    -   it can be found in brain, e.g. glial cells (e.g. astrocytes); in        the ovary; in the prostate, e.g. prostate epithelium; in tissues        undergoing large amounts of growth, differentiation and        angiogenesis, e.g. in the fetus and neonate (e.g. fetal and        neonatal kidney fetal heart and fetal adrenal gland); and in        cancerous tissue, e.g. tumors (e.g. Wilm's tumor, lung tumor,        colon tumor, metastases of colon tumor in the liver, metastases        of prostate tumor in the liver, and metastases of breast tumor        in the lung and brain);    -   it has a transfer domain which is preferably about 70%, 80%,        90%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid        residues about 280 to 411 of SEQ ID NO:2; and    -   it has a recognition/binding domain which is preferably about        70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to        amino acid residues about 30 to 250 of SEQ ID NO:2.

In a preferred embodiment the 25501 protein, or fragment thereof,differs from the corresponding sequence in SEQ ID NO:2. In oneembodiment it differs by at least one but by less than 15, 10 or 5 aminoacid residues. In another it differs from the corresponding sequence inSEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ IDNO:2. (If this comparison requires alignment the sequences should bealigned for maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.) The differencesare, preferably, differences or changes at a non-essential residue or aconservative substitution. In a preferred embodiment the differences arenot in the transfer domain at about residues 280 to 411 of SEQ ID NO:2.In another embodiment one or more differences are in the transfer domainat about residues 280 to 411 of SEQ ID NO:2.

Other embodiments include a protein that contains one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such 25501 proteins differ in amino acidsequence from SEQ ID NO:2, yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or morehomologous to SEQ ID NO:2. In another embodiment, the protein includesfragments or regions homologous to fragments, at least about 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or more homologous to a fragment of SEQ IDNO:2. A fragment of a 25501 protein can be at least 10, 15, 20, 25, 30,80, 130, 210, 300, or more amino acids in length. A fragment of a 25501protein can be a domain, e.g. a transfer domain or a fragment thereof(e.g. about amino acid residues 280 to 411, 280 to 320, 320 to 360 or360 to 411 of SEQ ID NO:2), or e.g. a recognition/binding domain or afragment thereof (e.g. about amino acid residues 30 to 250, 30 to 100,101 to 175 or 176 to 250 of SEQ ID NO:2). A fragment of a 25501 proteincan include the N-terminus or a fragment thereof (e.g. about amino acidresidues 1 to 30 of SEQ ID NO:2).

A 25501 protein or fragment is provided which varies from the sequenceof SEQ ID NO:2 in regions defined by amino acids about 1 to 279 or 412to 503 by at least one but by less than 15, 10 or 5 amino acid residuesin the protein or fragment but which does not differ from SEQ ID NO:2 inregions defined by amino acids about 280 to 411. (If this comparisonrequires alignment the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.) In some embodiments the difference is at anon-essential residue or is a conservative substitution, while in othersthe difference is at an essential residue or is a non-conservativesubstitution.

In one embodiment, a biologically active portion of a 25501 proteinincludes a transfer domain. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native 25501 protein.

In a preferred embodiment, the 25501 protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the 25501 protein issufficiently or substantially identical to SEQ ID NO:2. In yet anotherembodiment, the 25501 protein is sufficiently or substantially identicalto SEQ ID NO:2 and retains the functional activity of the protein of SEQID NO:2, as described in detail in the subsections above.

25501 Chimeric or Fusion Proteins

In another aspect, the invention provides 25501 chimeric or fusionproteins. As used herein, a 25501 “chimeric protein” or “fusion protein”includes a 25501 polypeptide linked to a non-25501 polypeptide. A“non-25501 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the 25501 protein, e.g., a protein which is different fromthe 25501 protein and which is derived from the same or a differentorganism. The 25501 polypeptide of the fusion protein can correspond toall or a portion e.g., a fragment described herein of a 25501 amino acidsequence. In a preferred embodiment, a 25501 fusion protein includes atleast one (or two) biologically active portion of a 25501 protein. Thenon-25501 polypeptide can be fused to the N-terminus or C-terminus ofthe 25501 polypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-25501 fusionprotein in which the 25501 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 25501. Alternatively, the fusion protein can be a 25501protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 25501 can be increased through use of a heterologous signalsequence.

Fusion proteins can include all or a part of a serum protein, e.g., aportion of an immunoglobulin (e.g., IgG, IgA, or IgE), e.g., an Fcregion and/or the hinge C1 and C2 sequences of an immunoglobulin orhuman serum albumin.

The 25501 fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. The25501 fusion proteins can be used to affect the bioavailability of a25501 substrate. 25501 fusion proteins can be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 25501 protein; (ii)mis-regulation of the 25501 gene; and (iii) aberrant post-translationalmodification of a 25501 protein.

Moreover, the 25501-fusion proteins of the invention can be used asimmunogens to produce anti-25501 antibodies in a subject, to purify25501 ligands and in screening assays to identify molecules whichinhibit the interaction of 25501 with a 25501 substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A 25501-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 25501 protein.

Variants of 25501 Proteins

In another aspect, the invention also features a variant of a 25501polypeptide, e.g., which functions as an agonist (mimetics) or as anantagonist. Variants of the 25501 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertion or deletion ofsequences or the truncation of a 25501 protein. An agonist of the 25501proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 25501protein. An antagonist of a 25501 protein can inhibit one or more of theactivities of the naturally occurring form of the 25501 protein by, forexample, competitively modulating a 25501-mediated activity of a 25501protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the25501 protein.

Variants of a 25501 protein can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of a 25501 protein foragonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 25501 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 25501 protein.

Variants in which a cysteine residues is added or deleted or in which aresidue which is glycosylated is added or deleted are particularlypreferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Recursiveensemble mutagenesis (REM), a new technique which enhances the frequencyof functional mutants in the libraries, can be used in combination withthe screening assays to identify 25501 variants (Arkin and Yourvan(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993)Protein Engineering 6:327-331).

Cell based assays can be exploited to analyze a variegated 25501library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 25501in a substrate-dependent manner. The transfected cells are thencontacted with 25501 and the effect of the expression of the mutant onsignaling by the 25501 substrate can be detected, e.g., by measuringtransferase activity, e.g., methyltransferase activity, e.g. the abilityto bind to, or interact with, a substrate or target molecule, e.g., anucleic acid molecule (e.g. DNA or RNA), a small organic molecule (e.g.,a hormone, a neurotransmitter or a coenzyme), or a protein; or theability to transfer a group, e.g. a methyl group from a donor to anacceptor molecule, e.g. the substrate or target molecule. Plasmid DNAcan then be recovered from the cells which score for inhibition, oralternatively, potentiation of signaling by the 25501 substrate, and theindividual clones further characterized.

In another aspect, the invention features a method of making a 25501polypeptide, e.g., a peptide having a non-wild type activity, e.g., anantagonist, agonist, or super agonist of a naturally occurring 25501polypeptide, e.g., a naturally occurring 25501 polypeptide. The methodincludes altering the sequence of a 25501 polypeptide, e.g., alteringthe sequence, e.g., by substitution or deletion of one or more residuesof a non-conserved region, a domain or residue disclosed herein, andtesting the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a 25501 polypeptide a biological activity of a naturallyoccurring 25501 polypeptide. The method includes altering the sequence,e.g., by substitution or deletion of one or more residues, of a 25501polypeptide, e.g., altering the sequence of a non-conserved region, or adomain or residue described herein, and testing the altered polypeptidefor the desired activity.

Anti-25501 Antibodies

In another aspect, the invention provides an anti-25501 antibody. Theterm “antibody” as used herein refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include scFV and dcFV fragments, Fab and F(ab)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspapain or pepsin, respectively.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., achimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

A full-length 25501 protein or, antigenic peptide fragment of 25501 canbe used as an immunogen or can be used to identify anti-25501 antibodiesmade with other immunogens, e.g., cells, membrane preparations, and thelike. The antigenic peptide of 25501 should include at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2 andencompasses an epitope of 25501. Preferably, the antigenic peptideincludes at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Fragments of 25501 which include residues about 121 to 132, from about150 to 160, and from about 410 to 423 of SEQ ID NO:2 can be used tomake, e.g., used as immunogens or used to characterize the specificityof an antibody, antibodies against hydrophilic regions of the 25501protein (see FIG. 1). Similarly, fragments of 25501 which includeresidues about amino acid 258 to 267, from about 353 to 363, and fromabout 100 to 108 of SEQ ID NO:2 can be used to make an antibody againsta hydrophobic region of the 25501 protein; fragments of 25501 whichinclude residues about 1 to 30, about 180 to 200, or about 490 to 503 ofSEQ ID NO:2 can be used to make an antibody against an intracellularregion of the 25501 protein; a fragment of 25501 which include residuesabout 280 to 411, about 300 to 320, or about 370 to 410 of SEQ ID NO:2can be used to make an antibody against the transfer region of the 25501protein; or a fragment of 25501 which include residues about 30 to 250,about 117 to 150, or about 151 to 198 of SEQ ID NO:2 can be used to makean antibody against the recognition/binding region of the 25501 protein.

Antibodies reactive with, or specific or selective for, any of theseregions, or other regions or domains described herein are provided.

Preferred epitopes encompassed by the antigenic peptide are regions of25501 located on the surface of the protein, e.g., hydrophilic regions,as well as regions with high antigenicity. For example, an Emini surfaceprobability analysis of the human 25501 protein sequence can be used toindicate the regions that have a particularly high probability of beinglocalized to the surface of the 25501 protein and are thus likely toconstitute surface residues useful for targeting antibody production.

In a preferred embodiment the antibody binds an epitope on any domain orregion on 25501 proteins described herein.

Additionally, chimeric, humanized, and completely human antibodies arealso within the scope of the invention. Chimeric, humanized, but mostpreferably, completely human antibodies are desirable for applicationswhich include repeated administration, e.g., therapeutic treatment ofhuman patients, and some diagnostic applications.

Chimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, can be made using standard recombinant DNAtechniques. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al. EuropeanPatent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559).

A humanized or complementarity determining region (CDR)-grafted antibodywill have at least one or two, but generally all three recipient CDR's(of heavy and or light immuoglobulin chains) replaced with a donor CDR.The antibody may be replaced with at least a portion of a non-human CDRor only some of the CDR's may be replaced with non-human CDR's. It isonly necessary to replace the number of CDR's required for binding ofthe humanized antibody to a 25501 or a fragment thereof. Preferably, thedonor will be a rodent antibody, e.g., a rat or mouse antibody, and therecipient will be a human framework or a human consensus framework.Typically, the immunoglobulin providing the CDR's is called the “donor”and the immunoglobulin providing the framework is called the “acceptor.”In one embodiment, the donor immunoglobulin is a non-human (e.g.,rodent). The acceptor framework is a naturally-occurring (e.g., a human)framework or a consensus framework, or a sequence about 85% or higher,preferably 90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, (1987) From Genesto Clones (Verlagsgesellschaft, Weinheim, Germany). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art. Humanizedantibodies can be generated by replacing sequences of the Fv variableregion which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison (1985) Science229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and by Queen etal. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents ofall of which are hereby incorporated by reference. Those methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of immunoglobulin Fv variable regions from at leastone of a heavy or light chain. Sources of such nucleic acid are wellknown to those skilled in the art and, for example, may be obtained froma hybridoma producing an antibody against a 25501 polypeptide orfragment thereof. The recombinant DNA encoding the humanized antibody,or fragment thereof, can then be cloned into an appropriate expressionvector.

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDR's of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534;Beidler et al. (1988) J. Immunol. 141:4053-4060; Winter U.S. Pat. No.5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Preferredhumanized antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, ahumanized antibody will have framework residues identical to the donorframework residue or to another amino acid other than the recipientframework residue. To generate such antibodies, a selected, small numberof acceptor framework residues of the humanized immunoglobulin chain canbe replaced by the corresponding donor amino acids. Preferred locationsof the substitutions include amino acid residues adjacent to the CDR, orwhich are capable of interacting with a CDR (see e.g., U.S. Pat. No.5,585,089). Criteria for selecting amino acids from the donor aredescribed in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat.No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, thecontents of which are hereby incorporated by reference. Other techniquesfor humanizing antibodies are described in Padlan et al. EP 519596 A1,published on Dec. 23, 1992.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), canbe engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

The anti-25501 antibody can be a single chain antibody. A single-chainantibody (scFV) can be engineered as described in, for example, Colcheret al. (1999) Ann. N Y Acad. Sci. 880:263-80; and Reiter (1996) Clin.Cancer Res. 2:245-52. The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target 25501 protein.

In a preferred embodiment, the antibody has reduced or no ability tobind an Fc receptor. For example, it is an isotype or subtype, fragmentor other mutant, which does not support binding to an Fc receptor, e.g.,it has a mutagenized or deleted Fc receptor binding region.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545)and analogs or homologs thereof. Therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine, vinblastine, taxol andmaytansinoids). Radioactive ions include, but are not limited to iodine,yttrium and praseodymium.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An anti-25501 antibody (e.g., monoclonal antibody) can be used toisolate 25501 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-25501 antibody can be used todetect 25501 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-25501 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labelling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In preferred embodiments, an antibody can be made by immunizing with apurified 25501 antigen, or a fragment thereof, e.g., a fragmentdescribed herein, tissues, e.g., crude tissue preparations, whole cells,preferably living cells, lysed cells, or cell fractions, e.g., cytosolicor nuclear fractions.

Antibodies which bind only a native 25501 protein, only denatured orotherwise non-native 25501 protein, or which bind both, are within theinvention. Antibodies with linear or conformational epitopes are withinthe invention. Conformational epitopes sometimes can be identified byidentifying antibodies which bind to native but not denatured 25501protein.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a 25501 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 25501 proteins,mutant forms of 25501 proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of 25501 proteins in prokaryotic or eukaryotic cells. Forexample, polypeptides of the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

Purified fusion proteins can be used in 25501 activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific or selective for 25501 proteins. In apreferred embodiment, a fusion protein expressed in a retroviralexpression vector of the present invention can be used to infect bonemarrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The 25501 expression vector can be a yeast expression vector, a vectorfor expression in insect cells, e.g., a baculovirus expression vector ora vector suitable for expression in mammalian cells.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub et al., (1986) Reviews—Trends inGenetics 1:1.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a 25501 nucleic acidmolecule within a recombinant expression vector or a 25501 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications canoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a25501 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovary(CHO) cells or CV-1 origin, SV-40 (COS) cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a25501 protein. Accordingly, the invention further provides methods forproducing a 25501 protein using the host cells of the invention. In oneembodiment, the method includes culturing the host cell of the invention(into which a recombinant expression vector encoding a 25501 protein hasbeen introduced) in a suitable medium such that a 25501 protein isproduced. In another embodiment, the method further includes isolating a25501 protein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 25501 transgene, or which otherwisemisexpress 25501. The cell preparation can consist of human or non-humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 25501transgene, e.g., a heterologous form of a 25501, e.g., a gene derivedfrom humans (in the case of a non-human cell). The 25501 transgene canbe misexpressed, e.g., overexpressed or underexpressed. In otherpreferred embodiments, the cell or cells include a gene whichmisexpresses an endogenous 25501, e.g., a gene the expression of whichis disrupted, e.g., a knockout. Such cells can serve as a model forstudying disorders which are related to mutated or misexpressed 25501alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 25501 polypeptide.

Also provided are cells, preferably human cells, e.g., humanhematopoietic or fibroblast cells, in which an endogenous 25501 is underthe control of a regulatory sequence that does not normally control theexpression of the endogenous 25501 gene. The expression characteristicsof an endogenous gene within a cell, e.g., a cell line or microorganism,can be modified by inserting a heterologous DNA regulatory element intothe genome of the cell such that the inserted regulatory element isoperably linked to the endogenous 25501 gene. For example, an endogenous25501 gene which is “transcriptionally silent,” e.g., not normallyexpressed, or expressed only at very low levels, can be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell. Techniquessuch as targeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a 25501 protein andfor identifying and/or evaluating modulators of 25501 activity. As usedherein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g:, a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 25501 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a 25501 protein toparticular cells. A transgenic founder animal can be identified basedupon the presence of a 25501 transgene in its genome and/or expressionof 25501 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding a25501 protein can further be bred to other transgenic animals carryingother transgenes.

25501 proteins or polypeptides can be expressed in transgenic animals orplants, e.g., a nucleic acid encoding the protein or polypeptide can beintroduced into the genome of an animal. In preferred embodiments thenucleic acid is placed under the control of a tissue specific promoter,e.g., a milk or egg specific promoter, and recovered from the milk oreggs produced by the animal. Suitable animals are mice, pigs, cows,goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed, e.g., below.

Uses

The nucleic acid molecules, proteins, protein homologs, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic).

The isolated nucleic acid molecules of the invention can be used, forexample, to express a 25501 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect a 25501mRNA (e.g., in a biological sample) or a genetic alteration in a 25501gene, and to modulate 25501 activity, as described further below. The25501 proteins can be used to treat disorders characterized byinsufficient or excessive production of a 25501 substrate or productionof 25501 inhibitors. In addition, the 25501 proteins can be used toscreen for naturally occurring 25501 substrates, to screen for drugs orcompounds which modulate 25501 activity, as well as to treat disorderscharacterized by insufficient or excessive production of 25501 proteinor production of 25501 protein forms which have decreased, aberrant orunwanted activity compared to 25501 wild type protein (e.g., aberrant ordeficient transferase, e.g. methyltransferase function or expression).Moreover, the anti-25501 antibodies of the invention can be used todetect and isolate 25501 proteins, regulate the bioavailability of 25501proteins, and modulate 25501 activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject 25501 polypeptide is provided. The methodincludes: contacting the compound with the subject 25501 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 25501 polypeptide. This method can beperformed in vitro, e.g., in a cell free system, or in vivo, e.g., in atwo-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules which interact with subject 25501polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 25501 polypeptide. Screening methods are discussed in moredetail below.

Screening Assays:

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to 25501 proteins, have astimulatory or inhibitory effect on, for example, 25501 expression or25501 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a 25501 substrate. Compounds thusidentified can be used to modulate the activity of target gene products(e.g., 25501 genes) in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a 25501 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a 25501 protein or polypeptideor a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994)J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-426; Zuckermann et al. (1994). J. Med. Chem. 37:2678-85; Cho etal. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233-51.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 25501 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate 25501 activity is determined. Determining the ability of thetest compound to modulate 25501 activity can be accomplished bymonitoring, for example, transferase, e.g. methyltransferase activitye.g. the binding to or interaction with a substrate or target molecule,e.g., a nucleic acid molecule (e.g. DNA or RNA), a small organicmolecule (e.g., a hormone, a neurotransmitter or a coenzyme), or aprotein or the transfer of a methyl group to or from the substrate ortarget molecule. The cell, for example, can be of mammalian origin,e.g., human.

The ability of the test compound to modulate 25501 binding to acompound, e.g., a 25501 substrate, or to bind to 25501 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to 25501 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 25501 could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulate25501 binding to a 25501 substrate in a complex. For example, compounds(e.g., 25501 substrates) can be labeled with ¹²⁵I, ¹⁴C, ³⁵S or ³H.,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., a 25501 substrate) to interact with25501 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 25501 without the labeling of either thecompound or the 25501. McConnell et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 25501.

In yet another embodiment, a cell-free assay is provided in which a25501 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to bind to the 25501protein or biologically active portion thereof is evaluated. Preferredbiologically active portions of the 25501 proteins to be used in assaysof the present invention include fragments which participate ininteractions with non-25501 molecules, e.g., fragments with high surfaceprobability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., 25501proteins or biologically active portions thereof) can be used in thecell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamnminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule can simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label can be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the 25501 protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander andUrbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA”detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIACORE™). Changes in the mass at the bindingsurface (indicative of a binding event) result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)), resulting in a detectable signal whichcan be used as an indication of real-time reactions between biologicalmolecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either 25501, an anti-25501 antibodyor its target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a25501 protein, or interaction of a 25501 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/25501 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione SEPHAROSE™ beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 25501 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 25501binding or activity determined using standard techniques.

Other techniques for immobilizing either a 25501 protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 25501 protein or target molecules can beprepared from biotin-NHS(N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific or selective for the immobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith 25501 protein or target molecules but which do not interfere withbinding of the 25501 protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or 25501protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the 25501 protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the 25501 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas andMinton (1993) Trends Biochem Sci 18:284-7); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. (1999) Current Protocols in MolecularBiology, J. Wiley, New York.); and immunoprecipitation (see, forexample, Ausubel et al., eds. (1999) Current Protocols in MolecularBiology, J. Wiley, New York). Such resins and chromatographic techniquesare known to one skilled in the art (see, e.g., Heegaard (1998) J MolRecognit 11: 141-8; Hage and Tweed (1997) J Chromatogr B Biomed SciAppl. 699:499-525). Further, fluorescence energy transfer can also beconveniently utilized, as described herein, to detect binding withoutfurther purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the 25501protein or biologically active portion thereof with a known compoundwhich binds 25501 to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a 25501 protein, wherein determining the ability of thetest compound to interact with a 25501 protein includes determining theability of the test compound to preferentially bind to 25501 orbiologically active portion thereof, or to modulate the activity of atarget molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are the25501 genes herein identified. In an alternative embodiment, theinvention provides methods for determining the ability of the testcompound to modulate the activity of a 25501 protein through modulationof the activity of a downstream effector of a 25501 target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined, or the binding of the effector to an appropriatetarget can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific or selective for thespecies to be anchored can be used to anchor the species to the solidsurface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific or selective for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific or selective for one of the bindingcomponents to anchor any complexes formed in solution, and a labeledantibody specific or selective for the other partner to detect anchoredcomplexes. Again, depending upon the order of addition of reactants tothe liquid phase, test compounds that inhibit complex or that disruptpreformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see; e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the 25501 proteins can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins, which bind to or interact with25501 (“25501-binding proteins” or “25501-bp”) and are involved in 25501activity. Such 25501-bps can be activators or inhibitors of signals bythe 25501 proteins or 25501 targets as, for example, downstream elementsof a 25501-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 25501 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GALA). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 25501 protein can bethe fused to the activator domain.) If the “bait” and the “prey”proteins are able to interact, in vivo, forming a 25501-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., lacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the 25501 protein.

In another embodiment, modulators of 25501 expression are identified.For example, a cell or cell free mixture is contacted with a candidatecompound and the expression of 25501 mRNA or protein evaluated relativeto the level of expression of 25501 mRNA or protein in the absence ofthe candidate compound. When expression of 25501 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of 25501 mRNA orprotein expression. Alternatively, when expression of 25501 mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 25501 mRNA or protein expression. Thelevel of 25501 mRNA or protein expression can be determined by methodsdescribed herein for detecting 25501 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 25501 protein can beconfirmed in vivo, e.g., in an animal such as an animal model foraberrant or deficient transferase, e.g. methyltransferase function orexpression.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 25501 modulating agent, an antisense 25501 nucleic acidmolecule, a 25501-specific antibody, or a 25501-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate25501 with a disease; (ii) identify an individual from a minutebiological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

Chromosome Mapping

The 25501 nucleotide sequences or portions thereof can be used to mapthe location of the 25501 genes on a chromosome. This process is calledchromosome mapping. Chromosome mapping is useful in correlating the25501 sequences with genes associated with disease.

Briefly, 25501 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the 25501 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 25501 sequences willyield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fanet al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map 25501 to achromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between a gene and adisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the 25501 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

25501 sequences can be used to identify individuals from biologicalsamples using, e.g., restriction fragment length polymorphism (RFLP). Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, the fragments separated, e.g., in a Southern blot,and probed to yield bands for identification. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the 25501 nucleotide sequences describedherein can be used to prepare two PCR primers from the 5′ and 3′ ends ofthe sequences. These primers can then be used to amplify an individual'sDNA and subsequently sequence it. Panels of corresponding DNA sequencesfrom individuals, prepared in this manner, can provide unique individualidentifications, as each individual will have a unique set of such DNAsequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3 are used, amore appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from 25501 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Use of Partial 25501 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 (e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 having a length of at least 20bases, preferably at least 30 bases) are particularly appropriate forthis use.

The 25501 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such 25501 probes can be used to identify tissue by speciesand/or by organ type.

In a similar fashion, these reagents, e.g., 25501 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofa gene which encodes 25501.

Such disorders include, e.g., a disorder associated with themisexpression of 25501 gene; a disorder of the nervous, apoptotic,angiogenic, cardiovascular, renal, immune, hepatic or reproductivesystem.

The method includes one or more of the following:

-   -   detecting, in a tissue of the subject, the presence or absence        of a mutation which affects the expression of the 25501 gene, or        detecting the presence or absence of a mutation in a region        which controls the expression of the gene, e.g., a mutation in        the 5′ control region;    -   detecting, in a tissue of the subject, the presence or absence        of a mutation which alters the structure of the 25501 gene;    -   detecting, in a tissue of the subject, the misexpression of the        25501 gene, at the mRNA level, e.g., detecting a non-wild type        level of an mRNA;    -   detecting, in a tissue of the subject, the misexpression of the        gene, at the protein level, e.g., detecting a non-wild type        level of a 25501 polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from the 25501gene; an insertion of one or more nucleotides into the gene, a pointmutation, e.g., a substitution of one or more nucleotides of the gene, agross chromosomal rearrangement of the gene, e.g., a translocation,inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:1, or naturally occurring mutants thereof or 5′ or 3′flanking sequences naturally associated with the 25501 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and detecting,by hybridization, e.g., in situ hybridization, of the probe/primer tothe nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 25501 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 25501.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a 25501 gene, an abnormal structure being indicative of risk for thedisorder.

In preferred embodiments the method includes contacting a sample fromthe subject with an antibody to the 25501 protein or a nucleic acid,which hybridizes specifically with the gene. These and other embodimentsare discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of 25501 protein or nucleic acid in abiological sample can be evaluated by obtaining a biological sample froma test subject and contacting the biological sample with a compound oran agent capable of detecting 25501 protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes 25501 protein such that the presence of 25501protein or nucleic acid is detected in the biological sample. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 25501 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 25501genes; measuring the amount of protein encoded by the 25501 genes; ormeasuring the activity of the protein encoded by the 25501 genes.

The level of mRNA corresponding to the 25501 gene in a cell can bedetermined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length 25501 nucleic acid, suchas the nucleic acid of SEQ ID NO:1, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to 25501 mRNA or genomic DNA. Other suitable probes for usein the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array. A skilled artisan canadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the 25501 genes.

The level of mRNA in a sample that is encoded by one of 25501 can beevaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987)U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al., (1989), Proc. Natl.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988)Biotechnology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. As used herein, amplification primers are defined asbeing a pair of nucleic acid molecules that can anneal to 5′ or 3′regions of a gene (plus and minus strands, respectively, or vice-versa)and contain a short region in between. In general, amplification primersare from about 10 to 30 nucleotides in length and flank a region fromabout 50 to 200 nucleotides in length. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule comprising the nucleotide sequence flanked by theprimers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the 25501gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting 25501 mRNA, or genomicDNA, and comparing the presence of 25501 mRNA or genomic DNA in thecontrol sample with the presence of 25501 mRNA or genomic DNA in thetest sample.

A variety of methods can be used to determine the level of proteinencoded by 25501. In general, these methods include contacting an agentthat selectively binds to the protein, such as an antibody with asample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

The detection methods can be used to detect 25501 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 25501 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 25501 protein include introducing into asubject a labeled anti-25501 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 25501protein, and comparing the presence of 25501 protein in the controlsample with the presence of 25501 protein in the test sample.

The invention also includes kits for detecting the presence of 25501 ina biological sample. For example, the kit can include a compound oragent capable of detecting 25501 protein or mRNA in a biological sample;and a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect 25501 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 25501 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

In one embodiment, a disease or disorder associated with aberrant orunwanted 25501 expression or activity is identified. A test sample isobtained from a subject and 25501 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 25501 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 25501 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted 25501 expression or activity. For example, such methods canbe used to determine whether a subject can be effectively treated withan agent for a transferase, e.g. methyltransferase disorder.

The methods of the invention can also be used to detect geneticalterations in a 25501 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in25501 protein activity or nucleic acid expression, such as atransferase, e.g. methyltransferase disorder. In preferred embodiments,the methods include detecting, in a sample from the subject, thepresence or absence of a genetic alteration characterized by at leastone of an alteration affecting the integrity of a gene encoding a25501-protein, or the mis-expression of the 25501 gene. For example,such genetic alterations can be detected by ascertaining the existenceof at least one of 1) a deletion of one or more nucleotides from a 25501gene; 2) an addition of one or more nucleotides to a 25501 gene; 3) asubstitution of one or more nucleotides of a 25501 gene, 4) achromosomal rearrangement of a 25501 gene; 5) an alteration in the levelof a messenger RNA transcript of a 25501 gene, 6) aberrant modificationof a 25501 gene, such as of the methylation pattern of the genomic DNA,7) the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a 25501 gene, 8) a non-wild type level of a 25501-protein,9) allelic loss of a 25501 gene, and 10) inappropriatepost-translational modification of a 25501-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 25501-gene. This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a 25501 gene under conditions such thathybridization and amplification of the 25501 gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.Alternatively, other amplification methods described herein or known inthe art can be used.

In another embodiment, mutations in a 25501 gene from a sample cell canbe identified by detecting alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined, e.g., by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 25501 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, twodimensional arrays, e.g., chip based arrays. Such arrays include aplurality of addresses, each of which is positionally distinguishablefrom the other. A different probe is located at each address of theplurality. The arrays can have a high density of addresses, e.g., cancontain hundreds or thousands of oligonucleotides probes (Cronin et al.(1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in 25501 can be identified intwo dimensional arrays containing light-generated DNA probes asdescribed in Cronin, M. T. et al. supra. Briefly, a first hybridizationarray of probes can be used to scan through long stretches of DNA in asample and control to identify base changes between the sequences bymaking linear arrays of sequential overlapping probes. This step allowsthe identification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the 25501 gene anddetect mutations by comparing the sequence of the sample 25501 with thecorresponding wild-type (control) sequence. Automated sequencingprocedures can be utilized when performing the diagnostic assays (Naeveet al. (1995) Biotechniques 19:448-53), including sequencing by massspectrometry.

Other methods for detecting mutations in the 25501 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242; Cotton et al. (1988) Proc. Natl. Acad Sci USA 85:4397; Saleebaet al. (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in 25501 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S.Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in 25501 genes. For example, single strandconformation polymorphism (SSCP) can be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control25501 nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments can belabeled or detected with labeled probes. The sensitivity of the assaycan be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al. (1986) Nature324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci USA 86:6230).

Alternatively, allele specific amplification technology which depends onselective PCR amplification can be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationcan carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification can also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189-93). Insuch cases, ligation will occur only if there is a perfect match at the3′ end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which can be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a 25501 gene.

Use of 25501 Molecules as Surrogate Markers

The 25501 molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 25501 molecules of the invention can be detected,and can be correlated with one or more biological states in vivo. Forexample, the 25501 molecules of the invention can serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers can serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease can be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection can be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The 25501 molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker can be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug can be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker can berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug can be sufficient to activate multiplerounds of marker (e.g., a 25501 marker) transcription or expression, theamplified marker can be in a quantity which is more readily detectablethan the drug itself. Also, the marker can be more easily detected dueto the nature of the marker itself; for example, using the methodsdescribed herein, anti-25501 antibodies can be employed in animmune-based detection system for a 25501 protein marker, or25501-specific radiolabeled probes can be used to detect a 25501 mRNAmarker. Furthermore, the use of a pharmacodynamic marker can offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The 25501 molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35:1650-1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, can be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 25501 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment can beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 25501 DNA can correlate with a 25501drug response. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-25501 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOR®EL solubilizer (BASF; Florham Park, N.J.) or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody, unconjugated or conjugated asdescribed herein, can include a single treatment or, preferably, caninclude a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent can, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted 25501expression or activity. As used herein, the term “treatment” is definedas the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

With regards to both prophylactic and therapeutic methods of treatment,such treatments can be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 25501 molecules ofthe present invention or 25501 modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwanted25501 expression or activity, by administering to the subject a 25501 oran agent which modulates 25501 expression or at least one 25501activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 25501 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 25501 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of25501 aberrance, for example, a 25501, 25501 agonist or 25501 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

It is possible that some 25501 disorders can be caused, at least inpart, by an abnormal level of gene product, or by the presence of a geneproduct exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

The 25501 molecules can act as novel diagnostic targets and/ortherapeutic agents for controlling one or more of neurologicaldisorders, cellular proliferative and/or differentiative disorders,ovary disorders, or prostate disorders, all of which are describedabove.

Aberrant or deficient activity or expression of transferases have beenassociated with, but are not limited to, apoptotic disorders, angiogenicdisorders, cardiovascular disorders, endothelial cell disorders, kidneydisorders, immune e.g., inflammatory, disorders, liver disorders, ormetabolic disorders, e.g., transferase-associated or other25501-associated disorders. As used herein, “transferase disorders” arediseases or disorders whose pathogeneses are caused by, is related to,or is associated with aberrant or deficient transferase protein functionor expression. Thus, the 25501 molecules can act as novel diagnostictargets and therapeutic agents for controlling one or more of apoptoticdisorders, angiogenic disorders, cardiovascular disorders, endothelialdisorders, kidney disorders, immune e.g., inflammatory, disorders, liverdisorders, or metabolic disorders.

The 25501 molecules can be used to treat apoptotic disorders. Disordersinvolving aberrant or deficient apoptosis include, but are not limitedto, autoimmune disorders such as systemic lupus erythematosus andimmune-mediated glomerulonephritis; neoplastic disorders such asfollicular lymphoma and hormone dependent tumors of the breast, prostategland and ovary; neurodegenerative disorders, such as Alzheimer'sdisease, Huntington's disease, retinitis pigmentosa, amyotrophic lateralsclerosis, spinal muscular atrophy and Parkinson's disease; viralinfections, such as those caused by herpesviruses, poxviruses andadenoviruses; blood disorders due to aberrant apoptotic activity in thebone marrow, such as anemia associated with chronic disease, i.e.,aplastic anemia, chronic neutropenia and myelodysplasia; and tissuedamage associated with myocardial infarctions and stroke.

The 25501 molecules can be used to treat angiogenic disorders. As usedherein, an “angiogenic disorder” includes a disease or disorder whichaffects or is caused by aberrant or deficient angiogenesis. Disordersinvolving angiogenesis include, but are not limited to, aberrant orexcess angiogenesis in tumors such as hemangiomas and Kaposi's sarcoma,von Hippel-Lindau disease, as well as the angiogenesis associated withtumor growth; aberrant or excess angiogenesis in diseases such as aCastleman's disease or fibrodysplasia ossificans progressiva; aberrantor deficient angiogenesis associated with aging, complications ofhealing certain wounds and complications of diseases such as diabetesand rheumatoid arthritis; or aberrant or deficient angiogenesisassociated with hereditary hemorrhagic telangiectasia, autosomaldominant polycystic kidney disease, myelodysplastic syndrome orKlippel-Trenaunay-Weber syndrome.

The 25501 molecules can be used to treat cardiovascular disorders. Asused herein, disorders involving the heart, or “cardiovascular disease”or a “cardiovascular disorder” includes a disease or disorder whichaffects the cardiovascular system, e.g., the heart, the blood vessels,and/or the blood. A cardiovascular disorder can be caused by animbalance in arterial pressure, a malfunction of the heart, or anocclusion of a blood vessel, e.g., by a thrombus. A cardiovasculardisorder includes, but is not limited to disorders such asarteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemiareperfusion injury, restenosis, arterial inflammation, vascular wallremodeling, ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, valvulardisease, including but not limited to, valvular degeneration caused bycalcification, rheumatic heart disease, endocarditis, or complicationsof artificial valves; atrial fibrillation, long-QT syndrome, congestiveheart failure, sinus node dysfunction, angina, heart failure,hypertension, atrial fibrillation, atrial flutter, pericardial disease,including but not limited to, pericardial effusion and pericarditis;cardiomyopathies, e.g., dilated cardiomyopathy or idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, ischemic disease, arrhythmia, sudden cardiac death, andcardiovascular developmental disorders (e.g., arteriovenousmalformations, arteriovenous fistulae, raynaud's syndrome, neurogenicthoracic outlet syndrome, causalgia/reflex sympathetic dystrophy,hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrialseptal defects, atrioventricular canal, coarctation of the aorta,ebsteins anomaly, hypoplastic left heart syndrome, interruption of theaortic arch, mitral valve prolapse, ductus arteriosus, patent foramenovale, partial anomalous pulmonary venous return, pulmonary atresia withventricular septal defect, pulmonary atresia without ventricular septaldefect, persistance of the fetal circulation, pulmonary valve stenosis,single ventricle, total anomalous pulmonary venous return, transpositionof the great vessels, tricuspid atresia, truncus arteriosus, ventricularseptal defects). A cardiovascular disease or disorder also can includean endothelial cell disorder.

The 25501 molecules can be used to treat endothelial cell disorders. Asused herein, an “endothelial cell disorder” includes a disordercharacterized by aberrant, unregulated, or unwanted endothelial cellactivity, e.g., proliferation, migration, angiogenesis, orvascularization; or aberrant expression of cell surface adhesionmolecules or genes associated with angiogenesis, e.g., TIE-2, FLT andFLK. Endothelial cell disorders include tumorigenesis, tumor metastasis,psoriasis, diabetic retinopathy, endometriosis, Grave's disease,ischemic disease (e.g., atherosclerosis), and chronic inflammatorydiseases (e.g., rheumatoid arthritis).

The 25501 molecules can be used to treat renal disorders. Disordersinvolving the kidney include, but are not limited to, congenitalanomalies including, but not limited to, cystic diseases of the kidney,that include but are not limited to, cystic renal dysplasia, polycystickidney diseases, and cystic diseases of renal medulla; glomerulardiseases including pathologies of glomerular injury that include, butare not limited to, in situ immune complex deposition, that includes,but is not limited to, anti-GBM nephritis, Heymann nephritis and othernephritis conditions, glomerulonephritis conditions, minimal changedisease (lipoid nephrosis), focal segmental glomerulosclerosis, IgAnephropathy (Berger disease); glomerular lesions associated withsystemic disease, including but not limited to, systemic lupuserythematosus, Henoch-Schonlein purpura, bacterial endocarditis,diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoidglomerulonephritis, and other systemic disorders; diseases affectingtubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, and other tubulointerstitialdiseases including, but not limited to, urate nephropathy, hypercalcemiaand nephrocalcinosis, and multiple myeloma; diseases of blood vesselsincluding benign nephrosclerosis, malignant hypertension and acceleratednephrosclerosis, renal artery stenosis, and thrombotic microangiopathiesincluding, but not limited to, hemolytic-uremic syndromes, and othervascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypemephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

The 25501 molecules can be used to treat adrenal disorders. Disordersinvolving the adrenal gland include, but are not limited to, congenitaladrenal hypoplasia, Addison's disease, Waterhouse-Friderichsen syndrome,Cushing's syndrome, Conn's syndrome, pheochromocytoma, or neuroblastoma.

The 25501 molecules can be used to treat immune, e.g., inflammatorydisorders. The 25501 nucleic acid and protein of the invention can beused to treat and/or diagnose a variety of immune, e.g., inflammatory,(e.g. respiratory inflammatory) disorders. Examples of immune disordersor diseases include, but are not limited to, autoimmune diseases(including, for example, diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, encephalomyelitis, myastheniagravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjögren's Syndrome, inflammatory bowel disease, e.g. Crohn's disease andulcerative colitis, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, asthma, allergic asthma, chronic obstructivepulmonary disease, cutaneous lupus erythematosus, scleroderma,vaginitis, proctitis, drug eruptions, leprosy reversal reactions,erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis), graft-versus-host disease, cases oftransplantation, and allergy such as, atopic allergy.

The 25501 molecules can be used to treat hepatic disorders. Disorderswhich can be treated or diagnosed by methods described herein include,but are not limited to, disorders associated with an accumulation in theliver of fibrous tissue, such as that resulting from an imbalancebetween production and degradation of the extracellular matrixaccompanied by the collapse and condensation of preexisting fibers. Themethods described herein can be used to diagnose or treat hepatocellularnecrosis or injury induced by a wide variety of agents includingprocesses which disturb homeostasis, such as an inflammatory process,tissue damage resulting from toxic injury or altered hepatic blood flow,and infections (e.g., bacterial, viral and parasitic). For example, themethods can be used for the early detection of hepatic injury, such asportal hypertension or hepatic fibrosis. In addition, the methods can beemployed to detect liver fibrosis attributed to inborn errors ofmetabolism, for example, fibrosis resulting from a storage disorder suchas Gaucher's disease (lipid abnormalities) or a glycogen storagedisease, A1-antitrypsin deficiency; a disorder mediating theaccumulation (e.g., storage) of an exogenous substance, for example,hemochromatosis (iron-overload syndrome) and copper storage diseases(Wilson's disease), disorders resulting in the accumulation of a toxicmetabolite (e.g., tyrosinemia, fructosemia and galactosemia) andperoxisomal disorders (e.g., Zellweger syndrome). Additionally, themethods described herein can be used for the early detection andtreatment of liver injury associated with the administration of variouschemicals or drugs, such as for example, methotrexate, isonizaid,oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, orwhich represents a hepatic manifestation of a vascular disorder such asobstruction of either the intrahepatic or extrahepatic bile flow or analteration in hepatic circulation resulting, for example, from chronicheart failure, veno-occlusive disease, portal vein thrombosis orBudd-Chiari syndrome.

The 25501 molecules can be used to treat metabolic disorders.Additionally, 25501 can play an important role in the regulation ofmetabolism. Diseases of metabolic imbalance include, but are not limitedto, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes.

As discussed, successful treatment of 25501 disorders can be broughtabout by techniques that serve to inhibit the expression or activity oftarget gene products. For example, compounds, e.g., an agent identifiedusing an assays described above, that proves to exhibit negativemodulatory activity, can be used in accordance with the invention toprevent and/or ameliorate symptoms of 25501 disorders. Such moleculescan include, but are not limited to peptides, phosphopeptides, smallorganic or inorganic molecules, or antibodies (including, for example,polyclonal, monoclonal, humanized, human, anti-idiotypic, chimeric orsingle chain antibodies, and Fab, F(ab′)₂ and Fab expression libraryfragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still further, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules can be utilized intreating or preventing a disease characterized by 25501 expression isthrough the use of aptamer molecules specific for 25501 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically or selectively bind to protein ligands(see, e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; andPatel (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid moleculescan in many cases be more conveniently introduced into target cells thantherapeutic protein molecules can be, aptamers offer a method by which25501 protein activity can be specifically decreased without theintroduction of drugs or other molecules which can have pluripotenteffects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiescan, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of 25501 disorders. For adescription of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject witha 25501 protein or epitope for stimulating antibody production isharmful to the subject, it is possible to generate an immune responseagainst 25501 through the use of anti-idiotypic antibodies (see, forexample, Herlyn (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee andFoon (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibodyis introduced into a mammal or human subject, it should stimulate theproduction of anti-anti-idiotypic antibodies, which should be specificto the 25501 protein. Vaccines directed to a disease characterized by25501 expression can also be generated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies can be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate 25501 disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays can utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate 25501 activity isused as a template, or “imprinting molecule”, to spatially organizepolymerizable monomers prior to their polymerization with catalyticreagents. The subsequent removal of the imprinted molecule leaves apolymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions. A detailed review of this technique can be seen inAnsell et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea(1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinitymatrixes are amenable to ligand-binding assays, whereby the immobilizedmonoclonal antibody component is replaced by an appropriately imprintedmatrix. An example of the use of such matrixes in this way can be seenin Vlatakis et al (1993) Nature 361:645-647. Through the use ofisotope-labeling, the “free” concentration of compound which modulatesthe expression or activity of 25501 can be readily monitored and used incalculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 25501expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 25501 or agent that modulates one or more ofthe activities of 25501 protein activity associated with the cell. Anagent that modulates 25501 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 25501 protein (e.g., a 25501 substrate orreceptor), a 25501 antibody, a 25501 agonist or antagonist, apeptidomimetic of a 25501 agonist or antagonist, or other smallmolecule.

In one embodiment, the agent stimulates one or 25501 activities.Examples of such stimulatory agents include active 25501 protein and anucleic acid molecule encoding 25501. In another embodiment, the agentinhibits one or more 25501 activities. Examples of such inhibitoryagents include antisense 25501 nucleic acid molecules, anti-25501antibodies, and 25501 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 25501 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g., upregulates or down regulates) 25501 expression or activity. In anotherembodiment, the method involves administering a 25501 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 25501 expression or activity.

Stimulation of 25501 activity is desirable in situations in which 25501is abnormally downregulated and/or in which increased 25501 activity islikely to have a beneficial effect. For example, stimulation of 25501activity is desirable in situations in which a 25501 is downregulatedand/or in which increased 25501 activity is likely to have a beneficialeffect. Likewise, inhibition of 25501 activity is desirable insituations in which 25501 is abnormally upregulated and/or in whichdecreased 25501 activity is likely to have a beneficial effect.

Pharmacogenomics

The 25501 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on 25501activity (e.g., 25501 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 25501-associated disorders (e.g.,aberrant or deficient transferase, e.g. methyltransferase function orexpression) associated with aberrant or unwanted 25501 activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) can be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician canconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a 25501 molecule or 25501modulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a 25501 molecule or 25501 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum et al. (1996)Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder et al. (1997) Clin.Chem. 43:254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare genetic defects or as naturally-occurringpolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP can occur once per every 1000 bases of DNA. ASNP can be involved in a disease process, however, the vast majority cannot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that can becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a 25501protein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a 25501 moleculeor 25501 modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a 25501 molecule or25501 modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 25501 genes of the present invention, wherein theseproducts can be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 25501genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent to which the unmodifiedtarget cells were resistant.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 25501 protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase 25501 gene expression, protein levels,or upregulate 25501 activity, can be monitored in clinical trials ofsubjects exhibiting decreased 25501 gene expression, protein levels, ordownregulated 25501 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease 25501 gene expression,protein levels, or downregulate 25501 activity, can be monitored inclinical trials of subjects exhibiting increased 25501 gene expression,protein levels, or upregulated 25501 activity. In such clinical trials,the expression or activity of a 25501 gene, and preferably, other genesthat have been implicated in, for example, a transferase-associated oranother 25501-associated disorder can be used as a “read out” or markersof the phenotype of a particular cell.

Other Embodiments

In another aspect, the invention features a method of analyzing aplurality of capture probes. The method is useful, e.g., to analyze geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence, wherein the capture probes are from acell or subject which expresses 25501 or from a cell or subject in whicha 25501 mediated response has been elicited; contacting the array with a25501 nucleic acid (preferably purified), a 25501 polypeptide(preferably purified), or an anti-25501 antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by a signal generated from a label attached to the25501 nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the 25501 nucleic acid, polypeptide,or antibody with a first array having a plurality of capture probes anda second array having a different plurality of capture probes. Theresults of each hybridization can be compared, e.g., to analyzedifferences in expression between a first and second sample. The firstplurality of capture probes can be from a control sample, e.g., a wildtype, normal, or non-diseased, non-stimulated, sample, e.g., abiological fluid, tissue, or cell sample. The second plurality ofcapture probes can be from an experimental sample, e.g., a mutant type,at risk, disease-state or disorder-state, or stimulated, sample, e.g., abiological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of 25501.Such methods can be used to diagnose a subject, e.g., to evaluate riskfor a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing 25501,e.g., analyzing structure, function, or relatedness to other nucleicacid or amino acid sequences. The method includes: providing a 25501nucleic acid or amino acid sequence; comparing the 25501 sequence withone or more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 25501.

The method can include evaluating the sequence identity between a 25501sequence and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.Preferred databases include GenBank™ and SwissProt.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles of25501. The set includes a plurality of oligonucleotides, each of whichhas a different nucleotide at an interrogation position, e.g., an SNP orthe site of a mutation. In a preferred embodiment, the oligonucleotidesof the plurality identical in sequence with one another (except fordifferences in length). The oligonucleotides can be provided withdifferential labels, such that an oligonucleotide which hybridizes toone allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

The sequences of 25501 molecules are provided in a variety of mediums tofacilitate use thereof. A sequence can be provided as a manufacture,other than an isolated nucleic acid or amino acid molecule, whichcontains a 25501 molecule. Such a manufacture can provide a nucleotideor amino acid sequence, e.g., an open reading frame, in a form whichallows examination of the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

A 25501 nucleotide or amino acid sequence can be recorded on computerreadable media. As used herein, “computer readable media” refers to anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as compact disc and CD-ROM; electrical storage media such asRAM, ROM, EPROM, EEPROM, and the like; and general hard disks andhybrids of these categories such as magnetic/optical storage media. Themedium is adapted or configured for having thereon 25501 sequenceinformation of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus of other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phones, pagers, and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe 25501 sequence information.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon a 25501nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

By providing the 25501 nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a transferase-associated or another 25501-associated disease ordisorder or a pre-disposition to a transferase-associated or another25501-associated disease or disorder, wherein the method comprises thesteps of determining 25501 sequence information associated with thesubject and based on the 25501 sequence information, determining whetherthe subject has a transferase-associated or another 25501-associateddisease or disorder and/or recommending a particular treatment for thedisease, disorder, or pre-disease condition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has atransferase-associated or another 25501-associated disease or disorderor a pre-disposition to a disease associated with 25501, wherein themethod comprises the steps of determining 25501 sequence informationassociated with the subject, and based on the 25501 sequenceinformation, determining whether the subject has atransferase-associated or another 25501-associated disease or disorderor a pre-disposition to a transferase-associated or another25501-associated disease or disorder, and/or recommending a particulartreatment for the disease, disorder, or pre-disease condition. Themethod may further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

The present invention also provides in a network, a method fordetermining whether a subject has a transferase-associated or another25501-associated disease or disorder or a pre-disposition to atransferase-associated or another 25501-associated disease or disorder,said method comprising the steps of receiving 25501 sequence informationfrom the subject and/or information related thereto, receivingphenotypic information associated with the subject, acquiringinformation from the network corresponding to 25501 and/or correspondingto a transferase-associated or another 25501-associated disease ordisorder, and based on one or more of the phenotypic information, the25501 information (e.g., sequence information and/or information relatedthereto), and the acquired information, determining whether the subjecthas a transferase-associated or another 25501-associated disease ordisorder or a pre-disposition to a transferase-associated or another25501-associated disease or disorder. The method may further comprisethe step of recommending a particular treatment for the disease,disorder, or pre-disease condition.

The present invention also provides a business method for determiningwhether a subject has a transferase-associated or another25501-associated disease or disorder or a pre-disposition to atransferase-associated or another 25501-associated disease or disorder,said method comprising the steps of receiving information related to25501 (e.g., sequence information and/or information related thereto),receiving phenotypic information associated with the subject, acquiringinformation from the network related to 25501 and/or related to atransferase-associated or another 25501-associated disease or disorder,and based on one or more of the phenotypic information, the 25501information, and the acquired information, determining whether thesubject has a transferase-associated or another 25501-associated diseaseor disorder or a pre-disposition to a transferase-associated or another25501-associated disease or disorder. The method may further comprisethe step of recommending a particular treatment for the disease,disorder, or pre-disease condition.

The invention also includes an array comprising a 25501 sequence of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be 25501. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative information, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue ifascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionin that tissue. Thus, one tissue can be perturbed and the effect on geneexpression in a second tissue can be determined. In this context, theeffect of one cell type on another cell type in response to a biologicalstimulus can be determined. In this context, the effect of one cell typeon another cell type in response to a biological stimulus can bedetermined. Such a determination is useful, for example, to know theeffect of cell-cell interaction at the level of gene expression. If anagent is administered therapeutically to treat one cell type but has anundesirable effect on another cell type, the invention provides an assayto determine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a transferase-associated or another 25501-associateddisease or disorder, progression of transferase-associated or another25501-associated disease or disorder, and processes, such a cellulartransformation associated with the transferase-associated or another25501-associated disease or disorder.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., acertaining the effect of 25501 expression on theexpression of other genes). This provides, for example, for a selectionof alternate molecular targets for therapeutic intervention if theultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 25501) that could serve asa molecular target for diagnosis or therapeutic intervention.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

Thus, the invention features a method of making a computer readablerecord of a sequence of a 25501 sequence which includes recording thesequence on a computer readable matrix. In a preferred embodiment therecord includes one or more of the following: identification of an ORF;identification of a domain, region, or site; identification of the startof transcription; identification of the transcription terminator; thefull length amino acid sequence of the protein, or a mature formthereof; the 5′ end of the translated region.

In another aspect, the invention features a method of analyzing asequence. The method includes: providing a 25501 sequence, or record, incomputer readable form; comparing a second sequence to the 25501sequence; thereby analyzing a sequence. Comparison can include comparingto sequences for sequence identity or determining if one sequence isincluded within the other, e.g., determining if the 25501 sequenceincludes a sequence being compared. In a preferred embodiment the 25501or second sequence is stored on a first computer, e.g., at a first siteand the comparison is performed, read, or recorded on a second computer,e.g., at a second site. E.g., the 25501 or second sequence can be storedin a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In apreferred embodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

This invention is further illustrated by the following exemplification,which should not be construed as limiting.

Exemplification

Gene Expression Analysis

TAQMAN™ quantitative PCR Total RNA was prepared from various humantissues by a single step extraction method using RNA STAT-60 accordingto the manufacturer's instructions (TelTest, Inc). Each RNA preparationwas treated with DNase I (Ambion) at 37° C. for 1 hour. DNAse Itreatment was determined to be complete if the sample required at least38 PCR amplification cycles to reach a threshold level of fluorescenceusing β-2 microglobulin as an internal amplicon reference. The integrityof the RNA samples following DNase I treatment was confirmed by agarosegel electrophoresis and ethidium bromide staining. After phenolextraction cDNA was prepared from the sample using the SUPERSCRIPT™Choice System following the manufacturer's instructions (GibcoBRL). Anegative control of RNA without reverse transcriptase was mock reversetranscribed for each RNA sample.

Human 25501 expression was measured by TAQMAN™ quantitative PCR (PerkinElmer Applied Biosystems) in cDNA prepared from a variety of normal anddiseased (e.g., cancerous) human tissues or cell lines.

Probes were designed by PrimerExpress software (PE Biosystems) based onthe sequence of the human 25501 gene. Each human 25501 gene probe waslabeled using FAM (6-carboxyfluorescein), and the β2-microglobulinreference probe was labeled with a different fluorescent dye, VIC. Thedifferential labeling of the target gene and internal reference genethus enabled measurement in same well. Forward and reverse primers andthe probes for both β2-microglobulin and target gene were added to theTAQMAN™ Universal PCR Master Mix (PE Applied Biosystems). Although thefinal concentration of primer and probe could vary, each was internallyconsistent within a given experiment. A typical experiment contained 200nM of forward and reverse primers plus 100 nM probe for β-2microglobulin and 600 nM forward and reverse primers plus 200 nM probefor the target gene. TaqMan matrix experiments were carried out on anABI PRISM™ 7700 Sequence Detection System (PE Applied Biosystems). Thethermal cycler conditions were as follows: hold for 2 min at 50° C. and10 min at 95° C., followed by two-step PCR for 40 cycles of 95° C. for15 sec followed by 60° C. for 1 min.

The following method was used to quantitatively calculate human 25501gene expression in the various tissues relative to β-2 microglobulinexpression in the same tissue. The threshold cycle (Ct) value is definedas the cycle at which a statistically significant increase influorescence is detected. A lower Ct value is indicative of a highermRNA concentration. The Ct value of the human 25501 gene is normalizedby subtracting the Ct value of the β-2 microglobulin gene to obtain a_(Δ)Ct value using the following formula:_(Δ)Ct=Ct_(human 25501)−Ct_(β-2 microglobulin). Expression is thencalibrated against a cDNA sample showing a comparatively low level ofexpression of the human 25501 gene. The _(Δ)Ct value for the calibratorsample is then subtracted from _(Δ)Ct for each tissue sample accordingto the following formula: _(ΔΔ)Ct=_(Δ)Ct-_(sample)−_(Δ)Ct-_(calibrator).Relative expression is then calculated using the arithmetic formulagiven by 2^(−ΔΔCt). Expression of the target human 25501 gene in each ofthe tissues tested is then graphically represented as discussed in moredetail below.

The results indicate significant 25501 expression in brain, e.g. glialcells (e.g. a high level in astrocytes); a medium level in the ovary; inthe prostate e.g. a medium level in prostate epithelium; in tissuesundergoing large amounts of growth, differentiation and angiogenesis,e.g. medium levels in the fetus and neonate (e.g. fetal and neonatalkidney fetal heart and fetal adrenal gland); and in cancerous tissue,e.g. tumors (e.g. medium levels in lung tumor, colon tumor andmetastases of colon tumor in the liver, and high levels in Wilm's tumorand metastases of prostate tumor in the liver).

Transcriptional Profiling The expression profiles of samples ofmetastatic brain and lung tumors originating from human breastadenocarcinoma tumors were compared with the profiles samples fromprimary human breast adenocarcinoma tumors. Total RNA was isolated fromthe tissue samples. Reverse transcriptase was used to generate³³P-dCTP-labeled cDNAs from the RNA. These experimental tissue cDNAswere hybridized to an array of molecules with known sequences. The nylonarray contained 9600 elements, each with a PCR product from cDNA clonesof the known genes. The hybridization levels from each tissue sample aremeasured and compared. Comparisons resulting in at least a 1.5-folddifference were judged as significant. The 25501 transcript wasidentified as being upregulated in the lung and brain metastatic tumorsoriginating from human breast adenocarcinoma tumors.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein.

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a nucleic acid molecule which encodes a polypeptide consisting of a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises the transfer domain of 25501 (amino acid residues 280 to 411 of SEQ ID NO:2) and has transferase activity; and c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, or a complement thereof, under conditions of 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. and the variant has transferase activity.
 2. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.
 3. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide.
 4. A host cell which contains the nucleic acid molecule of claim
 1. 5. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:2; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the variant is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, or a complement thereof, under conditions of 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. and the variant has transferase activity; and c) a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises the transfer domain of 25501, wherein the transfer domain of 25501 is amino acid residues 280 to 411 of SEQ ID NO:2, and the fragment has transferase activity.
 6. The isolated polypeptide of claim 5 comprising an amino acid sequence encoded by SEQ ID NO:1 or SEQ ID NO:3.
 7. The isolated polypeptide of claim 5c), wherein the fragment further comprises the recognition/binding domain of 25501, wherein the recognition/binding domain of 25501 is amino acid residues 30 to 250 of SEQ ID NO:2.
 8. The polypeptide of claim 5 further comprising heterologous amino acid sequences.
 9. An antibody which selectively binds to a polypeptide of claim
 5. 10. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, or a complement thereof, under conditions of 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. and the variant has transferase activity; and c) a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises the transfer domain of 25501, wherein the transfer domain of 25501 is amino acid residues 280 to 411 of SEQ ID NO:2, and the fragment has transferase activity; comprising culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.
 11. A method for detecting the presence of a polypeptide of claim 5 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 5; and b) determining whether the compound binds to the polypeptide in the sample.
 12. The method of claim 11, wherein the compound which binds to the polypeptide is an antibody.
 13. A kit comprising a compound which selectively binds to a polypeptide of claim 5 and instructions for use.
 14. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
 15. A method for identifying a compound which binds to a 25501 polypeptide comprising the steps of: a) contacting a 25501 polypeptide, or a cell expressing a 25501 polypeptide with a test compound; and b) determining whether the polypeptide binds to the test compound, wherein the 25501 polypeptide comprises a polypeptide selected from the group consisting of: i) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, ii) a polypeptide having the amino acid sequence of SEQ ID NO:2, iii) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises the transfer domain of 25501, wherein the transfer domain of 25501 is amino acid residues 280 to 411 of SEQ ID NO:2, and the fragment has transferase activity, iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2 and a non-25501 polypeptide, v) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises the transfer domain of 25501, wherein the transfer domain of 25501 is amino acid residues 280 to 411 of SEQ ID NO:2, and the fragment has transferase activity, and a non-25501 polypeptide, and vi) a fusion polypeptide encoded by a nucleic acid comprising a nucleotide sequence which encodes SEQ ID NO:2 operably linked to a non-25501 polypeptide.
 16. The method of claim 15, wherein the 25501 polypeptide is immobilized on a solid surface.
 17. The method of claim 15, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for 25501-transferase activity; and d) detection of binding using a two-hybrid assay.
 18. A method for modulating the activity of a polypeptide of claim 5 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 5 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
 19. A method for identifying a compound which modulates the activity of a polypeptide of claim 5, comprising: a) contacting a polypeptide of claim 5 or a cell expressing a polypeptide of claim 5 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
 20. The method of claim 19 wherein the cell is a tumor cell. 